Pullulanase Variants and Uses Thereof

ABSTRACT

A truncated pullulanase variant of a parent pullulanase belonging to family GH57 comprising an X47 domain and the use thereof.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to pullulanase variant having pullulanase activity and isolated polynucleotides encoding said pullulanase variants. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides. The invention also relates to the use of said pullulanase variants for starch convention processes include processes for producing a fermentation product, such as especially ethanol.

BACKGROUND OF THE INVENTION

A pullulanase is an enzyme which can degrade alpha-(1,6)-linkage of pullulan, amylopectin and other branched substrates. In the grain industry, bacterial pullulanases have been used for the purpose of removing alpha-1,6 bonds in starch, which may cause undesirable panose formation in the saccharification process.

Two different classes of pullulanases are known: EC 3.2.1.41 which include two types (type I and II) of pullulanases and EC 3.2.1.135 referred to as “neopullulanases”.

WO 95/23852 discloses an amylopullulanase from Thermococcus celer and the use for producing 5 sweeteners and ethanol from starch.

A pullulanase type II (family GH57) from the hyperthermophile Thermococcus hydrothermalis is disclosed as UNIPROT: Q9Y8I8.

A pullulanase type II (family GH57) from Thermococcus litoralis is disclosed in UNIPROT: Q8NKS8.

WO 98/26058 concerns a pullulanase from Thermococcus hydrothermalis CNCM 1 having a temperature optimum of 110° C. at pH 5.5 and the use thereof in combination with an alpha-amylase and alpha-glucosidase for producing syrups.

The object of the present invention is to provide pullulanases expressed in increased yields and/or having higher thermostability, respectively, compared to a parent pullulanase in question.

SUMMARY OF THE INVENTION

The present invention provides pullulanase variants of parent pullulanases belonging to family GH57 pullulanases.

In the first aspect the invention relates to pullulanase variants of parent pullulanases belonging to family GH57 and comprises an X47 domain, wherein the pullulanase variant is truncated at a position after the X47 domain.

In a preferred embodiment the parent family GH57 pullulanase may be derived from any bacterium. In a preferred embodiment the parent pullulanase is derived from a strain of the genus Thermococcus, preferably a strain of Thermococcus hydrothermalis, especially the mature part of SEQ ID NO: 2, or a strain of Thermococcus litoralis, especially the mature part of SEQ ID NO: 4 herein or the parent pullulanase is a hybrid pullulanase, e.g., comprising a sequence from Thermococcus hydrothermalis pullulanase and a sequence from Thermococcus litoralis pullulanase. Examples of parent pullulanases can be found below in the “Parent Pullulanases”-section.

More specifically the invention relates to pullulanase variants prepared from parent pullulanases belonging to family GH57 which comprises an X47 domain, wherein the parent pullulanase is the one shown in SEQ ID NO: 2 or 4 or 34, or another parent pullulanase having at least 60% identity to SEQ ID NOs: 2, 4 or 34, wherein the pullulanase variant comprises or consists of:

a) an amino acid sequence having pullulanase activity

-   -   i) having at least 60% identity to the sequence from amino acids         1-1009 of SEQ ID NO: 2, preferably to the sequence from amino         acids 1-782 of SEQ ID NO: 2; or     -   ii) having at least 60% identity to the sequence from amino         acids 1-988 of SEQ ID NO: 4, preferably to the sequence from         amino acids 1-781 of SEQ ID NO: 4;     -   iii) having at least 60% identity to the sequence from amino         acids 1-782 of SEQ ID NO: 34;

b) the parent pullulanase of SEQ ID NOs: 2, 4 or 34 is truncated at a position after the X47 domain;

c) another parent pullulanase having at least 60% identity to SEQ ID NOs: 2, 4 or 34 truncated in a position corresponding to the ones defined in a) or b);

d) a pullulanase variant defined in a), b) or c) having one or more (several) amino acids substituted, deleted, and/or inserted.

In another aspect the invention relates to an isolated polynucleotide encoding a pullulanase variant of the invention or an X47 domain of the invention selected from the group consisting of:

i) a polynucleotide having at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% identity with the pullulanase variant coding part of SEQ ID NOS: 1 or 3, or a complementary strand thereof;

ii) a polynucleotide having at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% identity with the X47 domain coding part of sequence SEQ ID NO: 1, 3, 37, or a complementary strand thereof; and

iii) a polynucleotide which hybridizes under medium stringency, preferably high stringency conditions with the pullulanase variant or X47 domain coding part of SEQ ID NO: 1, 3, or 37, or a complementary strand thereof.

The invention also relates to recombinant expression vectors comprising the nucleic acid construct of the invention and recombinant host cells comprising the nucleic acid construct of the invention or the vector of the invention.

In another aspect the invention relates to processes for producing a fermentation product from starch-containing material comprising the steps of:

(a) liquefying starch-containing material in the presence of an alpha-amylase and a family GH57 pullulanase;

(b) saccharifying the liquefied material obtained in step (a) using a carbohydrate-source generating enzyme;

(c) fermenting using a fermenting organism.

In a preferred embodiment the family GH57 pullulanase is a pullulanase variant of the invention.

In a further aspect the invention relates to the use of a family GH57 pullulanase or variants of the invention in a process of producing sweeteners from starch.

The invention also relates to the use of a family GH57 pullulanases or pullulanase variants of the invention in a process of producing a fermentation product, such as ethanol, from gelatinized and/or un-gelatinized starch.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an alignment of the mature part of the parent pullulanases from Thermococcus hydrothermalis (UNIPROT: Q9Y8I8) and Thermococcus litoralis (UNIPROT: Q8NKS8). Domains are annotated after CAZy (eFAM), GH57 (catalytic pullulanase domain), X47, DUF2223a and DUF2223b domains and linker (putative cell wall binding). Truncations X1, X4, X5 and X6 are indicated with left headed arrow.

FIG. 2 shows the truncation points X1, X4, X5 and X6 in the parent pullulanase derived from Thermococcus hydrothermalis (UNIPROT:Q9Y8I8).

FIG. 3 shows the HMM process used for identifying X47 domains.

FIG. 4 shows the temperature profiles of the purified pullulanase Thermococcus hydrothermalis (X1 truncation, expressed in Pichia pastoris), the truncation X4 of Thermococcus hydrothermalis and the hybrid (chimer) of Thermococcus hydrothermalis and Thermococcus litoralis (truncation site X4).

FIG. 5 shows the pH profiles of the purified pullulanases Thermococcus hydrothermalis (X1 truncation, expressed in Pichia pastoris), the truncation X4 of Thermococcus hydrothermalis and the hybrid (chimer) of Thermococcus hydrothermalis and Thermococcus litoralis (truncation site X4).

FIG. 6 shows HPLC quantification of ethanol titer (g/L) after 54 hours of SSF of mashes treated with increasing doses of Thermococcus hydrothermalis pullulanase at pH 5.4. The control did not have any pullulanase added during liquefaction. The columns represent the average of 5 replicate tubes while the error bars are the standard deviations of each treatment. The scale was 120 grams of total mash.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have prepared pullulanase variants of parent pullulanases belonging to family GH57 (Glycoside Hydrolase Family 57) comprising an X47 domain. A collection of GH57 pullulanases are described in Zone et al., 2004, Eur. J. Biochem. 271: 2863-2872 (incorporated by reference). However, in context of the invention GH57 pullulanases are not limited to those described in there. Generally Family GH57 is defined and updated by the CAZy-team and can be found on the CAZy-server (see cazy.org).

A parent pullulanase according to the invention belongs to family GH57 and is, preferably a pullulanase type II classified under EC 3.2.1.41. The specific parent pullulanases (UNIPROT: Q9Y8I8) and UNIPROT: Q8NKS8 used by the inventors are derived from strains of the hyperthermophile bacteria Thermococcus hydrothermalis and Thermococcus litoralis, respectively and hybrids thereof Several pullulanase variants were expressed in Bacillus subtilis and Pichia pastoria in several C-terminal truncated forms. For instance, a pullulanase variant truncated just after the X47 domain (truncation between 782 and 783 in SEQ ID NO: 2) showed a significant increased expression level and at the same time maintained pullulanase activity compared to the parent pullulanase.

Pullulanase Activity: Pullulanase activity means the ability to hydrolyze glycosidic alpha-(1,6)-linkages. It may be determined with pullulan or amylopectin as substrate, e.g., by the NPUN assay described below in the “Materials & Methods”-section or the AZCL-pullulan plates assay described in Example 4.

Variant: The term “variant” is defined herein as a polypeptide having pullulanase activity comprising an alteration, such as a substitution, insertion, deletion, truncation, of one or more (several) amino acid residues at one or more (several) specific positions of the mature parent pullulanase, e.g., of SEQ ID NO: 2, 4 or 34. The altered polynucleotide is obtained through human intervention by modification of the polynucleotide sequence disclosed, e.g., in SEQ ID NO: 1 or 3; or a homologous sequence thereof.

The pullulanase variant of the present invention may have at least 20%, at least 40%, at least 50%, at least 60%, at least 70%, least 80%, at least 90%, at least 95%, or at least 100% of the pullulanase activity of the mature parent pullulanase, such as the parent pullulanase shown in SEQ ID NOS: 2, 4, or 34.

Wild-Type Enzyme: The term “wild-type” pullulanase denotes a pullulanase expressed by a naturally occurring microorganism, such as a bacterium, yeast, or filamentous fungus found in nature.

Parent Enzyme: The term “parent” pullulanase as used herein means a pullulanase to which a modification, e.g., substitution(s), insertion(s), deletion(s), and/or truncation(s), is made to produce the pullulanase variants of the present invention. This term also refers to the pullulanase with which a variant is compared and aligned. The parent may be a naturally occurring (wild-type) pullulanase or a variant. For instance, the parent pullulanase may be a variant of a naturally occurring pullulanase which has been modified or altered in the amino acid sequence. A parent may also be an allelic variant, which is a pullulanase encoded by any of two or more alternative forms of a gene occupying the same chromosomal locus.

Isolated variant or polypeptide: The term “isolated variant” or “isolated pullulanase” as used herein refers to a variant or a pullulanase that is isolated from a source. In one aspect, the pullulanase variant or pullulanase is at least 1% pure, preferably at least 5% pure, more preferably at least 10% pure, more preferably at least 20% pure, more preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, and most preferably at least 90% pure, as determined by SDS-PAGE.

Substantially pure variant or polypeptide: The term “substantially pure variant” or “substantially pure pullulanase” denotes herein a polypeptide preparation that contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polypeptide material with which it is natively or recombinantly associated. It is, therefore, preferred that the substantially pure variant or polypeptide is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99%, most preferably at least 99.5% pure, and even most preferably 100% pure by weight of the total polypeptide material present in the preparation. The variants and polypeptides of the present invention are preferably in a substantially pure form. This can be accomplished, for example, by preparing the variant or polypeptide by well-known recombinant methods or by classical purification methods.

Mature polypeptide: The term “mature pullulanase” is defined herein as a polypeptide having pullulanase activity that is in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one preferred embodiment, the mature pullulanase is amino acids 1 to 1310 of SEQ ID NO: 2 and amino acids 1-1065 for SEQ ID NO: 4. Amino acids -1 to -27 of SEQ ID NO: 2 and amino acids -1 to -24 of SEQ ID NO: 4 are signal peptides.

Mature polypeptide coding sequence: The term “mature polypeptide coding sequence” is defined herein as a nucleotide sequence that encodes a mature pullulanase. In one embodiment, the mature pullulanase coding sequence is nucleotides 82 to 4011 of SEQ ID NO: 1 and nucleotide 73-3267 of SEQ ID NO: 3. Nucleotides 1 to 81 of SEQ ID NO: 1 and nucleotides 1-72 of SEQ ID NO: 3 encode signal peptides.

Alignment: Alignment of two amino acid sequence in order to identify corresponding position is according to the invention done by using the MUSCLE (Multiple Sequence Comparison by Log-Expectation) alignment program (Edgar, Robert C. (2004), MUSCLE: multiple sequence alignment with high accuracy and high throughput, Nucleic Acids Research 32(5), 1792-97.) with 16 iterations of the protein sequence alignments.

Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “identity”. For purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16: 276-277; emboss.org), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the—nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment)

For purposes of the present invention, the degree of identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra; emboss.org), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the—nobrief option) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps in Alignment)

Allelic variant: The term “allelic variant” denotes herein any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

Isolated polynucleotide: The term “isolated polynucleotide” as used herein refers to a polynucleotide that is isolated from a source. In one aspect, the isolated polynucleotide is at least 1% pure, preferably at least 5% pure, more preferably at least 10% pure, more preferably at least 20% pure, more preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, and most preferably at least 90% pure, and even most preferably at least 95% pure, as determined by agarose electrophoresis.

Substantially pure polynucleotide: The term “substantially pure polynucleotide” as used herein refers to a polynucleotide preparation free of other extraneous or unwanted nucleotides and in a form suitable for use within genetically engineered polypeptide production systems. Thus, a substantially pure polynucleotide contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polynucleotide material with which it is natively or recombinantly associated. A substantially pure polynucleotide may, however, include naturally occurring 5′ and 3′ untranslated regions, such as promoters and terminators. It is preferred that the substantially pure polynucleotide is at least 90% pure, preferably at least 92% pure, more preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, even more preferably at least 98% pure, most preferably at least 99%, and even most preferably at least 99.5% pure by weight. The polynucleotides of the present invention are preferably in a substantially pure form, i.e., that the polynucleotide preparation is essentially free of other polynucleotide material with which it is natively or recombinantly associated. The polynucleotides may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.

Coding sequence: When used herein the term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of its polypeptide product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA, synthetic, or recombinant polynucleotide.

Nucleic acid construct: The term “nucleic acid construct” as used herein refers to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic. The term nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present invention.

Control sequences: The term “control sequences” is defined herein to include all components necessary for the expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence may be native or foreign to the polynucleotide encoding the polypeptide or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.

Operably linked: The term “operably linked” denotes herein a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence such that the control sequence directs the expression of the coding sequence of a polypeptide.

Expression: The term “expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector: The term “expression vector” is defined herein as a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide of the present invention and is operably linked to additional nucleotides that provide for its expression.

Host cell: The term “host cell”, as used herein, includes any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

Hybridization: The polynucleotide may be able to hybridize with the mature polypeptide coding sequence of SEQ ID NOs: 1 or 3 or any other polynucleotide encoding a mature family GH57 pullulanase. The hybridization may be done by prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and either 25% formamide (for very low and low stringencies), 35% formamide (for medium and medium-high stringencies), or 50% formamide (for high and very high stringencies), following standard Southern blotting procedures for 12 to 24 hours optimally. The carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS preferably at 45° C. (very low stringency), 50° C. (low stringency), 55° C. (medium stringency), 60° C. (medium-high stringency), 65° C. (high stringency), or 70° C. (very high stringency).

Parent Pullulanases

Parent pullulanases according to the invention are family GH57 pullulanases which having an X47 domain. The pullulanases are classified under EC 3.2.1.41 and are referred to as pullulanase type II or sometimes “amylopullulanases”. Type II pullulanases are in contrast to type I pullulanases, which specifically attack alpha-1,6 linkages, also able to hydrolyze alpha-1,4 linkages.

In a preferred embodiment the parent pullulanase belongs to family GH57 and comprises an X47 domain and optional DUF2223a and/or DUF2223b domains. Domains and families can be found in the Pfam protein families database: Finn et al., 2008, Nucleic Acids Research Database Issue 36: D281-D288. The Pfam database is a collection of protein families, each represented by multiple sequence alignments and Hidden Markov Models (HMMs). The parent family GH57 pullulanase may be obtained from any source, such as a microorganism, preferably a bacterium or fungal organism, such as yeast and a filamentous fungus. In a preferred embodiment the parent pullulanase is a wild-type enzyme. In a preferred embodiment the parent pullulanase is derived from a bacterium, preferably of the genus Thermococcus or Pyrococcus, including the ones in the table below.

Thermococcus hydrothermalis. SWISSPROT: Q9Y8I8 Thermococcus sp. HJ21. SWISSPROT: B6SED6 Thermococcus onnurineus SWISSPROT: B6YV54 (strain NA1). Thermococcus kodakaraensis. SWISSPROT: Q5JJ55 Thermococcus sp. AM4. SWISSPROT: B7QZQ4 Pyrococcus furiosus. SWISSPROT: Q8TZQ1 Pyrococcus furiosus DSM 3638. SWISSPROT: Q3HUR3 Pyrococcus furiosus. SWISSPROT: O30772 Thermococcus gammatolerans SWISSPROT: C5A4E3 (strain DSM 15229/JCM 11827/EJ3). Thermococcus barophilus MP. SWISSPROT: B5IRL5 Thermococcus litoralis. SWISSPROT: Q8NKS8 Pyrococcus abyssi. SWISSPROT: Q9V294

In a preferred embodiment the parent pullulanase is derived from a strain from the genus Thermococcus or Pyrococcus, including Thermococcus litoralis, preferably the mature part of SEQ ID NO: 4 or Thermococcus hydrothermalis, preferably the mature part of SEQ ID NO: 2. A parent pullulanase may also be a hybrid (chimeric) enzyme, preferably between bacterial pullulanases, especially the catalytic domain from one pullulanase and the X47 domain from another pullulanase. In a preferred embodiment the parent pullulanase comprises two Thermococcus pullulanases, preferably the catalytic domain of Thermococcus hydrothermalis and the X47 domain of Thermococcus litoralis, especially the chimeric pullulanase shown in SEQ ID NO: 34. The parent pullulanase may have at least 85%, preferably at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the mature parent pullulanase of above mentioned pullulanase, preferably the mature part of the parent pullulanases shown in SEQ ID NO: 2, 4 or 34. The total number of different amino acids in the parent pullulanase shown in SEQ ID NO: 2, 4 or 34, or another parent pullulanase may be fifteen, more preferably fourteen, even more preferably thirteen, even more preferably twelve, even more preferably eleven, even more preferably ten, even more preferably nine, even more preferably eight, even more preferably seven, even more preferably six, even more preferably five, even more preferably four, even more preferably three, even more preferably two, and most preferably one.

The parent pullulanase is encoded by a nucleic acid sequence which may hybridize under medium, more preferably high stringency conditions, with the nucleic acid sequence of SEQ ID NO: 1 or 3, or its complementary strand.

Family GH57 Enzymes and GH57 Domains

Family GH57 enzymes, including Family GH57 pullulanases (EC 3.2.1.41), are defined by the CAZy-team headed by Bernard Henrissat (Architecture et Fonction des Macromolécules Biologiques UMR6098, CNRS/Université de Provence/Université de la Méditerranée, Parc Scientifique et Technologique de Luminy Case 932 163 Avenue de Luminy 13288 Marseille Cedex 09, France). An updated list of sequences belonging to the family GH57 can be found on the CAZy-server (cazy.org). Zone et al., 2004, Eur. J. Biochem. 271: 2863-2872 (incorporated by reference) collected 59 amino acid sequence belonging to family GH57 GH57 domains) from glycoside hydrolases using the CAZy server, Pfam database and BLAST tools including the Thermococcus hydrothermalis pullulanase sequence (Q9Y8i18_THEHY) and the Thermococcus litoralis pullulanase sequence (Q8NKS8). These family GH57 proteins/domains are incorporated by reference. Previous work by Erra-Pujada et al. (“The type II pullulanase of Thermococcus hydrothermalis: molecular characterization of the gene and expression of the catalytic domain”. J Bacteriology 181(10): 3284-3287 (1999)) lead to the GH57 domain prediction as being the catalytic core of the enzyme. According to the invention the parent pullulanase belongs to the family GH57 (Glycoside Hydrolase Family 57). Examples of GH57 domains include the amino acid sequence from 156-436 of SEQ ID NO: 2, amino acid sequence 156-436 of SEQ ID NO: 4 and amino acid sequence 156-436 of SEQ ID NO: 34 (see also FIG. 1). The GH57 domain is located N-terminal to the X47 domain in the parent pullulanase. In an embodiment of the invention the family GH57 pullulanase comprises a GH57 domain and an X47 domain. The GH57 domain may be a domain having at least 60% identity, preferably at least 70%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identity to the amino acid sequences from 156-436 of SEQ ID NO: 2, amino acid sequence 156-436 of SEQ ID NO: 4, or amino acid sequence 156-436 of SEQ ID NO: 34.

X47 Domains

An X47 domain is a domain found downstream from the GH57 domain in a family GH57 pullulanase as defined above. X47 domain may be identified using a Hidden Markov Model (HMM). Examples of X47 domains are shown in SEQ ID NOs: 20-30. In an embodiment the X47 domain is a domain having at least 60% identity, preferably at least 70%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identity to the amino acid sequence from 580-768 of SEQ ID NO: 2 or the amino acid sequence 579-767 of SEQ ID NO: 4, or amino acids 86-274 in SEQ ID NO: 38, or to any of SEQ ID NOs: 20-30.

DUF2223a and DUF2223b Domains

As mentioned above the parent pullulanase may comprise DUF2223 domains which have no known function. The DUF2223a and DUF2223b domains are located C-terminal to the X47 domain (see FIG. 1). The DUF2223 members, as of release 24, 13. Oct. 2009, are found in various prokaryotic membrane-anchored proteins predicted to be involved in the regulation of pullulanases (pfam.sanger.ac.uk/family/DUF2223).

Pullulanase Variants of the Invention

In the first aspect the invention relates to a pullulanase variant of a parent pullulanase belonging to family GH57 (comprising a GH57 domain) comprising an X47 domain, wherein the pullulanase is truncated after the X47 domain. Examples of GH57 domains and X47 domains are mentioned above. The variant may be truncated after the X47 domain or just before the end of the X47 domain (such as 1-10 amino acids), i.e., in the X47 domain. However, it is preferred to truncate after the X47 domain. Examples of parent pullulanase are mentioned above in the “Parent Pullulanases”-section. A pullulanase variant of the invention may have one or more (several) amino acids substituted, deleted, and/or inserted compared to the parent pullulanase in SEQ ID NO: 2, 4 or 34, or another parent pullulanase. The pullulanase variant may be at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 85%, more preferably 90%, more preferably 95%, more preferably 97%, at least 98%, at least 99% identical to the mature parent pullulanase, preferably the parent pullulanases shown in SEQ ID NOs: 2, 4, or 34. The total number of different amino acids in the pullulanase variant compared to the mature parent pullulanase may be fifteen, more preferably fourteen, even more preferably thirteen, even more preferably twelve, even more preferably eleven, even more preferably ten, even more preferably nine, even more preferably eight, even more preferably seven, even more preferably six, even more preferably five, even more preferably four, even more preferably three, even more preferably two, and most preferably one. It is to be understood that a pullulanase variant of the invention has pullulanase activity. In an embodiment the truncation is in the DUF2223a domain or DUF223b domain. According to the invention the truncation is within 100 amino acids, preferably 50 amino acids, preferably 20 amino acids after the end of the X47 domain.

In a specific and preferred embodiment the pullulanase variant of the invention is a variant prepared from a parent pullulanase belonging to family GH57 which comprises an X47 domain, wherein the parent pullulanase is the one shown in SEQ ID NO: 2, 4 or 34, or another parent pullulanase having at least 60% identity to SEQ ID NO: 2, 4, or 34, wherein the pullulanase variant comprises or consists of:

a) An Amino Acid Sequence having Pullulanase Activity;

-   -   i) having at least 60% identity to the sequence from amino acids         1-1009 of SEQ ID NO: 2, preferably to the sequence from amino         acids 1-782 of SEQ ID NO: 2; or     -   ii) having at least 60% identity to the sequence from amino         acids 1-988 of SEQ ID NO: 4, preferably to the sequence from         amino acids 1-781 of SEQ ID NO: 4;     -   iii) having at least 60% identity to the sequence from amino         acids 1-782 of SEQ ID NO: 34;

b) the parent pullulanase of SEQ ID NOS: 2, 4, or 34 is truncated at a position after the X47 domain;

c) another parent pullulanase having at least 60% identity to SEQ ID NOs: 2, 4 or 34 which is truncated in a position corresponding to the ones defined in a) or b);

d) a pullulanase variant defined in a), b) or c) having one or more (several) amino acids substituted, deleted, and/or inserted.

In a preferred embodiment the pullulanase variant has at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the mature part of the pullulanases in SEQ ID NOS: 2, 4 or 34, preferably the sequence from amino acids 1-1009 of SEQ ID NO: 2 or from amino acids 1-987 in SEQ ID NO: 4 (X2 truncation), preferably to the sequence from amino acids 1-782 (X4 truncation) of SEQ ID NOS: 2 or from the sequence from amino acids 1-781 of SEQ ID NO: 4 (X4 truncation) or amino acids 1-782 SEQ ID NO: 34 (X4 truncation). In an embodiment the parent pullulanase is a wild-type pullulanase. In an embodiment the truncation is in the DUF2223a domain located from positions 769-1009 in SEQ ID NO: 2, which corresponds to positions 768-988 in SEQ ID NO: 4, or in a corresponding positions in another parent pullulanase. In a preferred embodiment the truncation is between amino acids in positions 782-783 in SEQ ID NO: 2 (X4 truncation), which corresponds to positions between positions 781-782 in SEQ ID NO: 4,or in corresponding positions in another parent pullulanase. According to the invention the truncation is typically within 100 amino acids, preferably 50 amino acids, preferably 20 amino acids of the end of the X47 domain, which ends at position 768 in SEQ ID NO: 2 and position 767 in SEQ ID NO: 4 and 768 in SEQ ID NO: 34, or a corresponding position in another parent pullulanase. A variant of the invention may have higher pullulanase activity compared to the parent pullulanase. In an embodiment the variant has improved thermostability compared to the corresponding parent pullulanase, especially the parent pullulanase shown in SEQ ID NOS: 2, 4 or 34. The pullulanase variant may have a temperature optimum in the range between 65-100° C., preferably 70-90° C., especially 75-85° C.; and/or may have a pH optimum in the range between pH 40-6.

X47 Domain of the Invention

The present invention also relates to X47 domains. The X47 may be obtainable from a parent pullulanase as exemplified above including from a strain from the genus Thermococcus, including Thermococcus sp. AM4, Thermococcus sp. HJ21, Thermococcus barophilus, Thermococcus gammatolerans, Thermococcus kodakarensis, Thermococcus litoralis; Thermococcus hydrothermalis; Thermococcus onnurineus; or obtained from a strain of the genus Pyrococcus, such as Pyrococcus abyssi and Pyrococcus furiosus. In an embodiment the X47 domain comprising or consisting of the amino acid sequence 580-768 in SEQ ID NO: 2 or amino acid sequence 579-767 in SEQ ID NO: 4 or amino acids 580-768 in SEQ ID NO: 34, or amino acids 86-274 in SEQ ID NO: 38, or a corresponding position in another parent pullulanases. In an embodiment an X47 domain of the invention comprising or consisting of the amino acid sequence 580-768 in SEQ ID NO: 2 or amino acid sequence 579-767 in SEQ ID NO: 4 or amino acids 580-768 in SEQ ID NO: 34, or the amino acid sequence 86-274 in SEQ ID NO: 38, or a corresponding positions in another parent pullulanases determined by Hidden Markov Model (hmm) having a score of at least 300, preferably a score of 350, preferably a score of 400, preferably between 300-500, such as between 380-450. In an embodiment the X47 domain is an amino acid sequence having at least 60% identity to amino acid sequence 580-768 in SEQ ID NO: 2 or to amino acid sequence 579-767 in SEQ ID NO: 4 or amino acid sequence 580-768 in SEQ ID NO: 34, or the amino acid sequence 86-274 in SEQ ID NO: 38. In a preferred embodiment the X47 has at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence 580-768 in SEQ ID NO: 2 or to amino acid sequence 579-767 in SEQ ID NO: 4 or amino acid sequence 580-768 in SEQ ID NO: 34, or the amino acid sequence 86-274 in SEQ ID NO: 38.

An Isolated Polynucleotide Encoding a Pullulanase Variant or X47 Domain

In an embodiment the invention related to an isolated polynucleotide encoding a pullulanase variant of the invention or an X47 domain of the invention selected from the group consisting of:

i) a polynucleotide having at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% identity with the pullulanase variant coding part of SEQ ID NOS: 1 or 3, or a complementary strand thereof;

ii) a polynucleotide having at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% identity with the X47 domain coding part of sequence SEQ ID NO: 1, 3, or 37, or a complementary strand thereof; and

iii) a polynucleotide which hybridizes under medium stringency, preferably high stringency conditions with the pullulanase variant or X47 domain coding part of SEQ ID NO: 1, 3, or 37, or a complementary strand thereof.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprising the polynucleotide described above, operably linked to one or more (several) control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. The control sequence may include an appropriate promoter sequence, a nucleotide sequence that is recognized by a host cell for expression of a polynucleotide encoding a pullulanase variant or X47 domain of the present invention. The promoter sequence contains transcriptional control sequences that mediate the expression of the pullulanase variant or X47 domain. The promoter may be any nucleotide sequence that shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. The control sequence may also include a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3′ terminus of the nucleotide sequence encoding the pullulanase variant or X47 domain in question. Any terminator that is functional in the host cell of choice may be used in the present invention. The control sequence may also include a suitable leader sequence, a nontranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5′ terminus of the nucleotide sequence encoding the pullulanase variant or X47 domain of the invention. Any leader sequence that is functional in the host cell of choice may be used in the present invention. The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3′ terminus of the nucleotide sequence and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell of choice may be used in the present invention. The control sequence may also include a signal peptide coding sequence that codes for an amino acid sequence linked to the amino terminus of a pullulanase variant or X47 domain and directs the encoded pullulanase variant or X47 domain into the cell's secretory pathway. The 5′ end of the coding sequence of the nucleotide sequence may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the secreted polypeptide in question. Alternatively, the 5′ end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. The foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, the foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide in question. However, any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell of choice, i.e., secreted into a culture medium, may be used in the present invention. The control sequence may also include a propeptide coding sequence that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide in question. It may also be desirable to add regulatory sequences that allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Examples of useful control sequences are described in WO 2007/090402.

Expression Vectors

The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleic acids and control sequences described herein may be joined together to produce a recombinant expression vector that may include one or more (several) convenient restriction sites to allow for insertion or substitution of the nucleotide sequence encoding the polypeptide at such sites. Alternatively, a polynucleotide sequence of the present invention may be expressed by inserting the nucleotide sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression. Examples of vector systems are described in WO 2007/090402.

Host Cells

The present invention also relates to recombinant host cells, comprising an isolated polynucleotide of the present invention, which are advantageously used in the recombinant production of the polypeptides. A vector comprising a polynucleotide of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source. Examples of host cells are described in WO 2007/090402.

Methods of Production

The present invention also relates to methods of producing a pullulanase variants or X47 domain of the present invention, comprising: (a) cultivating a cell, which in its wild-type form produces the polypeptide in question, under conditions conducive for production of the polypeptide in question; and (b) recovering the polypeptide in question. The present invention also relates to methods of producing a pullulanase variant or X47 domain of the present invention, comprising: (a) cultivating a recombinant host cell, as described herein, under conditions conducive for production of the polypeptide in question; and (b) recovering the polypeptide in question. The expression level of a pullulanase variant of the invention may according to the invention be the same or higher compared to the corresponding parent pullulanase under the same conditions. The production may be carried out as described in WO 2007/090402.

Processes for Producing Fermentation Products from Un-Gelatinized Starch-Containing Material

In this aspect the invention relates to processes for producing fermentation products from starch-containing material without gelatinization (i.e., without cooking) of the starch-containing material. According to the invention the desired fermentation product, such as ethanol, can be produced without liquefying the aqueous slurry containing the starch-containing material and water. In one embodiment a process of the invention includes saccharifying (e.g., milled) starch-containing material, e.g., granular starch, below the initial gelatinization temperature, preferably in the presence of alpha-amylase and/or carbohydrate-source generating enzyme(s) to produce sugars that can be fermented into the desired fermentation product by a suitable fermenting organism. In this embodiment the desired fermentation product, preferably ethanol, is produced from un-gelatinized (i.e., uncooked), preferably milled, cereal grains, such as corn. Accordingly, in the first aspect the invention relates to processes for producing fermentation products from starch-containing material comprising simultaneously saccharifying and fermenting starch-containing material using a carbohydrate-source generating enzyme and a fermenting organism at a temperature below the initial gelatinization temperature of said starch-containing material in the presence of a pullulanase variant of the invention. In an embodiment a protease is also present. The protease may be any acid fungal amylase or metallo protease. Examples are listed below in the “Protease” section. The fermentation product, such as especially ethanol, may optionally be recovered after fermentation, e.g., by distillation. Suitable starch-containing starting materials are listed in the “Starch-Containing Materials”-section below. Contemplated enzymes are listed in the “Enzymes”-section below. Typically amylase(s), such as glucoamylase(s) and/or other carbohydrate-source generating enzymes, and/or alpha-amylase(s), is(are) present during fermentation. Examples of glucoamylases and other carbohydrate-source generating enzymes can be found below and includes raw starch hydrolyzing glucoamylases. Examples of alpha-amylase(s) include acid alpha-amylases, preferably acid fungal alpha-amylases. Examples of fermenting organisms include yeast, preferably a strain of Saccharomyces cerevisiae. Other suitable fermenting organisms are listed in the “Fermenting Organisms”-section above. The term “initial gelatinization temperature” means the lowest temperature at which starch gelatinization commences. In general, starch heated in water begins to gelatinize between about 50° C. and 75° C.; the exact temperature of gelatinization depends on the specific starch and can readily be determined by the skilled artisan. Thus, the initial gelatinization temperature may vary according to the plant species, to the particular variety of the plant species as well as with the growth conditions. In context of this invention the initial gelatinization temperature of a given starch-containing material may be determined as the temperature at which birefringence is lost in 5% of the starch granules using the method described by Gorinstein et al., 1992, Starch/Stärke 44(12): 461-466. Before initiating the process a slurry of starch-containing material, such as granular starch, having 10-55 w/w-% dry solids (DS), preferably 25-45 w/w-% dry solids, more preferably 30-40 w/w-% dry solids of starch-containing material may be prepared. The slurry may include water and/or process waters, such as stillage (backset), scrubber water, evaporator condensate or distillate, side-stripper water from distillation, or process water from other fermentation product plants. Because the process of the invention is carried out below the initial gelatinization temperature, and thus no significant viscosity increase takes place, high levels of stillage may be used if desired. In an embodiment the aqueous slurry contains from about 1 to about 70 vol.-%, preferably 15-60 vol.-%, especially from about 30 to 50 vol.-% water and/or process waters, such as stillage (backset), scrubber water, evaporator condensate or distillate, side-stripper water from distillation, or process water from other fermentation product plants, or combinations thereof, or the like. The starch-containing material may be prepared by reducing the particle size, preferably by dry or wet milling, to 0.05 to 3.0 mm, preferably 0.1-0.5 mm. After being subjected to a process of the invention at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or preferably at least 99% of the dry solids in the starch-containing material are converted into a soluble starch hydrolyzate. A process in this aspect of the invention is conducted at a temperature below the initial gelatinization temperature, which means that the temperature typically lies in the range between 30-75° C., preferably between 45-60° C. In a preferred embodiment the process carried at a temperature from 25° C. to 40° C., such as from 28° C. to 35° C., such as from 30° C. to 34° C., preferably around 32° C. In an embodiment the process is carried out so that the sugar level, such as glucose level, is kept at a low level, such as below 6 w/w-%, such as below about 3 w/w-%, such as below about 2 w/w-%, such as below about 1 w/w-%., such as below about 0.5 w/w-%, or below 0.25 w/w-%, such as below about 0.1 w/w-%. Such low levels of sugar can be accomplished by simply employing adjusted quantities of enzyme and fermenting organism. A skilled person in the art can easily determine which doses/quantities of enzyme and fermenting organism to use. The employed quantities of enzyme and fermenting organism may also be selected to maintain low concentrations of maltose in the fermentation broth. For instance, the maltose level may be kept below about 0.5 w/w-%, such as below about 0.2 w/w-%. The process of the invention may be carried out at a pH from about 3 and 7, preferably from pH 3.5 to 6, or more preferably from pH 4 to 5. In an embodiment fermentation is ongoing for 6 to 120 hours, in particular 24 to 96 hours.

Processes for Producing Fermentation Products from Gelatinized Starch-Containing Material

In this aspect the invention relates to processes for producing fermentation products, especially ethanol, from starch-containing material, which process includes a liquefaction step and sequentially or simultaneously performed saccharification and fermentation steps. Consequently, the invention relates to processes for producing fermentation products from starch-containing material comprising the steps of:

(a) liquefying starch-containing material in the presence of an alpha-amylase and a family GH57 pullulanase or;

(b) saccharifying the liquefied material obtained in step (a) using a carbohydrate-source generating enzyme;

(c) fermenting using a fermenting organism.

Examples of family GH57 pullulanasess can be found above. In a preferred embodiment the pullulanase is a pullulanase variant of the invention.

In an embodiment a protease, such as an acid fungal protease or a metallo protease is added before, during and/or after liquefaction. The protease may be any of the ones mentioned below in the “Protease”-section. In a preferred embodiment the metallo protease is derived from a strain of the genus Thermoascus, preferably a strain of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670. The alpha-amylase may be any of the ones mentioned in the “Alpha-Amylase”-section below. In a preferred embodiment the alpha-amylase is a fungal alpha-amylase, preferably derived from the genus Aspergillus, especially a strain of A. niger, A. oryzae, A. awamori, or A. kawachii, or of the genus Rhizomucor, preferably a strain the Rhizomucor pusillus, or the genus Meripilus, preferably a strain of Meripilus giganteus or the alpha-amylase disclosed in Richardson et al., 2002, The Journal of Biological Chemistry 277(29): 267501-26507 (Issue 19 July), referred to as BD5088. The carbohydrate-source generating enzymes may be any of the ones mentioned below in the Carbohydrate-Source Generating Enzyme“-section. In a preferred embodiment the carbohydrate-source generating enzyme is a glucoamylase derived from a strain of Aspergillus, preferably Aspergillus niger or Aspergillus awamori, a strain of Talaromyces, especially Talaromyces emersonii; or a strain of Athelia, especially Athelia rolfsii; a strain of Trametes, preferably Trametes cingulata; a strain of the genus Pachykytospora, preferably a strain of Pachykytospora papyracea; or a strain of the genus Leucopaxillus, preferably Leucopaxillus giganteus; or a strain of the genus Peniophora, preferably a strain of the species Peniophora rufomarginata; or a mixture thereof. Saccharification step (b) and fermentation step (c) may be carried out either sequentially or simultaneously. The pullulanase variant and/or metallo protease may be added during saccharification and/or fermentation when the process is carried out as a sequential saccharification and fermentation process and before or during fermentation when steps (b) and (c) are carried out simultaneously (SSF process). The pullulanase variant and/or metallo protease may also advantageously be added before liquefaction (pre-liquefaction treatment), i.e., before or during step (a), and/or after liquefaction (post liquefaction treatment), i.e., after step (a). The pullulanase variant is most advantageously added before or during liquefaction, i.e., before or during step (a). The fermentation product, such as especially ethanol, may optionally be recovered after fermentation, e.g., by distillation. Suitable starch-containing starting materials are listed in the section “Starch-Containing Materials”-section below. Contemplated enzymes are listed in the “Enzymes”-section below. The liquefaction is preferably carried out in the presence of at least an alpha-amylase, preferably a bacterial alpha-amylase or acid fungal alpha-amylase. The fermenting organism is preferably yeast, preferably a strain of Saccharomyces cerevisiae. Suitable fermenting organisms are listed in the “Fermenting Organisms”-section below.

In a particular embodiment, the process of the invention further comprises, prior to step (a), the steps of:

x) reducing the particle size of the starch-containing material, preferably by milling (e.g., using a hammer mill);

y) forming a slurry comprising the starch-containing material and water.

In a preferred embodiment the particle size is smaller than a #7 screen, preferably a #6 screen. A #7 screen is usually used in conventional prior art processes. The aqueous slurry may contain from 10-55 w/w-% dry solids (DS), preferably 25-45 w/w-% dry solids (DS), more preferably 30-40 w/w-% dry solids (DS) of starch-containing material. The slurry is heated to above the gelatinization temperature and alpha-amylase, preferably bacterial and/or acid fungal alpha-amylase may be added to initiate liquefaction (thinning). The slurry may in an embodiment be jet-cooked to further gelatinize the slurry before being subjected to alpha-amylase in step (a). Liquefaction may in an embodiment be carried out as a three-step hot slurry process. The slurry is heated to between 60-95° C., preferably between 70-90° C., such as preferably between 80-85° C. at pH 4-6, preferably 4.5-5.5, and alpha-amylase, together with pullulanase variant and/or protease, preferably metallo protease, are added to initiate liquefaction (thinning). In an embodiment the slurry may then be jet-cooked at a temperature between 95-140° C., preferably 100-135° C., such as 105-125° C., for about 1-15 minutes, preferably for about 3-10 minutes, especially around about 5 minutes. The slurry is cooled to 60-95° C. and more alpha-amylase and optionally pullulanase variant and/or protease, preferably metallo protease, is(are) added to finalize hydrolysis (secondary liquefaction). The liquefaction process is usually carried out at pH 4.0-6, in particular at a pH from 4.5 to 5.5. Saccharification step (b) may be carried out using conditions well-known in the art. For instance, a full saccharification process may last up to from about 24 to about 72 hours, however, it is common only to do a pre-saccharification of typically 40-90 minutes at a temperature between 30-65° C., typically about 60° C., followed by complete saccharification during fermentation in a simultaneous saccharification and fermentation process (SSF process). Saccharification is typically carried out at temperatures from 20-75° C., preferably from 40-70° C., typically around 60° C., and at a pH between 4 and 5, normally at about pH 4.5. The most widely used process in fermentation product, especially ethanol, production is the simultaneous saccharification and fermentation (SSF) process, in which there is no holding stage for the saccharification, meaning that fermenting organism, such as yeast, and enzyme(s), may be added together. SSF may typically be carried out at a temperature from 25° C. to 40° C., such as from 28° C. to 35° C., such as from 30° C. to 34° C., preferably around about 32° C. In an embodiment fermentation is ongoing for 6 to 120 hours, in particular 24 to 96 hours.

Fermentation Medium

“Fermentation media” or “fermentation medium” refers to the environment in which fermentation is carried out and which includes the fermentation substrate, that is, the carbohydrate source that is metabolized by the fermenting organism. The fermentation medium may comprise nutrients and growth stimulator(s) for the fermenting organism(s). Nutrient and growth stimulators are widely used in the art of fermentation and include nitrogen sources, such as ammonia; urea, vitamins and minerals, or combinations thereof.

Fermenting Organisms

The term “fermenting organism” refers to any organism, including bacterial and fungal organisms, suitable for use in a fermentation process and capable of producing the desired fermentation product. Especially suitable fermenting organisms are able to ferment, i.e., convert, sugars, such as glucose or maltose, directly or indirectly into the desired fermentation product. Examples of fermenting organisms include fungal organisms, such as yeast. Preferred yeast includes strains of Saccharomyces spp., in particular, Saccharomyces cerevisiae. In one embodiment the fermenting organism is added to the fermentation medium so that the viable fermenting organism, such as yeast, count per mL of fermentation medium is in the range from 10⁵ to 10¹², preferably from 10⁷ to 10¹⁰, especially about 5×10⁷. Commercially available yeast includes, e.g., RED START™ and ETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI (available from Fleischmann's Yeast, USA), SUPERSTART and THERMOSACC™ fresh yeast (available from Ethanol Technology, WI, USA), BIOFERM AFT and XR (available from NABC—North American Bioproducts Corporation, GA, USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL (available from DSM Specialties).

Starch-Containing Materials

Any suitable starch-containing material may be used according to the present invention. The starting material is generally selected based on the desired fermentation product. Examples of starch-containing materials, suitable for use in a process of the invention, include whole grains, corn, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum, rice, peas, beans, or sweet potatoes, or mixtures thereof or starches derived there from, or cereals. Contemplated are also waxy and non-waxy types of corn and barley. The term “granular starch” means raw uncooked starch, i.e., starch in its natural form found in cereal, tubers or grains. Starch is formed within plant cells as tiny granules insoluble in water. When put in cold water, the starch granules may absorb a small amount of the liquid and swell. At temperatures up to 50° C. to 75° C. the swelling may be reversible. However, with higher temperatures an irreversible swelling called “gelatinization” begins. Granular starch to be processed may be a highly refined starch quality, preferably at least 90%, at least 95%, at least 97% or at least 99.5% pure or it may be a more crude starch-containing materials comprising (e.g., milled) whole grains including non-starch fractions such as germ residues and fibers. The raw material, such as whole grains, may be reduced in particle size, e.g., by milling, in order to open up the structure and allowing for further processing. Two processes are preferred according to the invention: wet and dry milling. In dry milling whole kernels are milled and used. Wet milling gives a good separation of germ and meal (starch granules and protein) and is often applied at locations where the starch hydrolysate is used in production of, e.g., syrups. Both dry and wet milling is well known in the art of starch processing and is equally contemplated for a process of the invention. In an embodiment the particle size is reduced to between 0.05 to 3.0 mm, preferably 0.1-0.5 mm, or so that at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90% of the starch-containing material fit through a sieve with a 0.05 to 3.0 mm screen, preferably 0.1-0.5 mm screen.

Fermentation Products

The term “fermentation product” means a product produced by a process including a fermentation step using a fermenting organism. Fermentation products contemplated according to the invention include alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H₂ and CO₂); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B₁₂, beta-carotene); and hormones. In a preferred embodiment the fermentation product is ethanol, e.g., fuel ethanol; drinking ethanol, i.e., potable neutral spirits; or industrial ethanol or products used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry and tobacco industry. Preferred beer types comprise ales, stouts, porters, lagers, bitters, malt liquors, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer. Preferred fermentation processes used include alcohol fermentation processes. The fermentation product, such as ethanol, obtained according to the invention, may preferably be used as fuel. However, in the case of ethanol it may also be used as potable ethanol.

Recovery

Subsequent to fermentation the fermentation product may be separated from the fermentation medium. The slurry may be distilled to extract the desired fermentation product or the desired fermentation product may be extracted from the fermentation medium by micro or membrane filtration techniques. Alternatively the fermentation product may be recovered by stripping. Methods for recovery are well known in the art.

Use of Pullulanase Variants

A pullulanase variant of the invention may be used in the conversion of starch for the production of dextrose, syrup (such as high-fructose syrup), edible products (such as snack pellets), ethanol or beer, e.g., as described in WO 2000/001796, WO 2001/051620, WO 2006/213132, or WO 2003/024242. Thus, it may be used in the liquefaction of starch (WO 2006/028897), in beer brewing (WO 2007/144393), or for saccharification in combination with a glucoamylase (EP 63909).

Enzymes Proteases

According to the present invention the protease used may be of any origin. In a preferred embodiment the protease may be an acid fungal protease or a metallo protease. In a preferred embodiment the protease is a metalloprotease derived from a strain of the genus Thermoascus, preferably a strain of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670 disclosed in WO 2003/048353 (Novozymes). According to the invention a peptidase and other protein degrading enzymes are referred to as proteases. In a preferred embodiment the protease is an endo-protease and/or an exo-protease. Suitable proteases may be of fungal, bacterial, including filamentous fungi and yeast, and plant origin. In an embodiment the protease is an acidic protease, i.e., a protease characterized by the ability to hydrolyze proteins under acidic conditions below pH 7, e.g., at a pH between 2-7. In an embodiment the acidic protease has an optimum pH in the range from 2.5 and 3.5 (determined on high nitrogen casein substrate at 0.7% w/v at 37° C.) and a temperature optimum between 5 to 50° C. at an enzyme concentration of 10 mg/mL at 30° C. for one hour in 0.1 M piperazine/acetate/glycine buffer). In another embodiment the protease is an alkaline protease, i.e., a protease characterized by the ability to hydrolyze proteins under alkaline conditions above pH 7, e.g., at a pH between 7 and 11. In an embodiment the alkaline protease is derived from a strain of Bacillus, preferably Bacillus licheniformis. In an embodiment the alkaline protease has an optimum temperature in the range from 7 and 11 and a temperature optimum around 70° C. determined at pH 9. In another embodiment the protease is a neutral protease, i.e., a protease characterized by the ability to hydrolyze proteins under conditions between pH 5 and 8. In an embodiment the alkaline protease is derived from a strain of Bacillus, preferably Bacillus amyloliguefaciens. In an embodiment the alkaline protease has an optimum pH in the range between 7 and 11 (determined at 25° C., 10 minutes reaction time with an enzyme concentration of 0.01-0.2 AU/L) and a temperature optimum between 50° C. and 70° C. (determined at pH 8.5, 10 minutes reaction time and 0.03-0.3 AU/L enzyme concentration. In an embodiment the protease is a metallo protease. In a preferred embodiment the protease is derived from a strain of the genus Thermoascus, preferably a strain of Thermoascus aurantiacus, especially Thermoaccus aurantiacus CGMCC No. 0670 having the sequence shown in the mature part of SEQ ID NO: 2 in WO 2003/048353 hereby incorporated by reference. The Thermoaccus aurantiacus protease is active from 20-90° C., with an optimum temperature around 70° C. Further, the enzyme is activity between pH 5-10 with an optimum around pH 6. Suitable plant proteases may be derived from barley. Suitable bacterial proteases include Bacillus proteases derived from Bacillus amyloliguefaciens and Bacillus licheniformis. Suitable filamentous bacterial proteases may be derived from a strain of Nocardiopsis, preferably Nocardiopsis prasina NRRL 18262 protease (or Nocardiopsis sp. 10R) and Nocardiopsis dassonavilla NRRL 18133 (Nocardiopsis dassonavilla M58-1) both described in WO 88/03947 (Novozymes). Suitable acid fungal proteases include fungal proteases derived from Aspergillus, Mucor, Rhizomucor, Rhizopus, Candida, Coriolus, Endothia, Enthomophtra, Irpex, Penicillium, Sclerotium, Thermoaccus, and Torulopsis. Especially contemplated are proteases derived from Aspergillus niger (see, e.g., Koaze et al., 1964, Agr. Biol. Chem. Japan 28, 216), Aspergillus saitoi (see, e.g., Yoshida, 1954, J. Agr. Chem. Soc. Japan 28: 66), Aspergillus awamori (Hayashida et al., 1977, Agric. Biol. Chem. 42(5): 927-933, Aspergillus aculeatus (WO 95/02044), or Aspergillus oryzae; proteases from Mucor pusillus or Mucor miehei disclosed in U.S. Pat. No. 4,357,357 and U.S. Pat. No. 3,988,207; and Rhizomucor mehei or Rhizomucor pusillus disclosed in, e.g., WO 94/24880 (hereby incorporated by reference). Aspartic acid proteases are described in, for example, Hand-book of Proteolytic Enzymes, Edited by A. J. Barrett, N. D. Rawlings and J. F. Woessner, Aca-demic Press, San Diego, 1998, Chapter 270). Suitable examples of aspartic acid protease include, e.g., those disclosed in Berka et al., 1990, Gene 96: 313; Berka et al., 1993, Gene 125: 195-198; and Gomi et al., 1993, Biosci. Biotech. Biochem. 57: 1095-1100, which are hereby incorporated by reference. Commercially available products include ALCALASE®, ESPERASE™, NEUTRASE®, RENILASE®, NOVOZYM™ FM 2.0L, and NOVOZYM™ 50006 (available from Novozymes A/S, Denmark) and GC106™ and SPEZYME™ FAN from Genencor Int., Inc., USA. The protease may be present in concentrations in the range from 0.0001 to 1.0 wt.-% of TS, preferably 0.001 to 0.1 wt.-% of TS.

Alpha-Amylase

According to the invention any alpha-amylase may be used, such as of fungal, bacterial or plant origin. In a preferred embodiment the alpha-amylase is an acid alpha-amylase, e.g., acid fungal alpha-amylase or acid bacterial alpha-amylase. The term “acid alpha-amylase” means an alpha-amylase (E.C. 3.2.1.1) which added in an effective amount has activity optimum at a pH in the range of 3 to 7, preferably from 3.5 to 6, or more preferably from 4-5.

Bacterial Alpha-Amylase

According to the invention a bacterial alpha-amylase is preferably derived from the genus Bacillus. In a preferred embodiment the Bacillus alpha-amylase is derived from a strain of Bacillus licheniformis, Bacillus amyloliguefaciens, Bacillus subtilis or Bacillus stearothermophilus, but may also be derived from other Bacillus sp. Specific examples of contemplated alpha-amylases include the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 4 in WO 99/19467, the Bacillus amyloliguefaciens alpha-amylase SEQ ID NO: 5 in WO 99/19467 and the Bacillus stearothermophilus alpha-amylase shown in SEQ ID NO: 3 in WO 99/19467 (all sequences hereby incorporated by reference). In an embodiment the alpha-amylase may be an enzyme having a degree of identity of at least 60%, preferably at least 70%, more preferred at least 80%, even more preferred at least 90%, such as at least 95%, at least 96%, at least 97%, at least 98% or at least 99% to any of the sequences shown in SEQ ID NOS: 1, 2 or 3, respectively, in WO 99/19467. The Bacillus alpha-amylase may also be a variant and/or hybrid, especially one described in any of WO 96/23873, WO 96/23874, WO 97/41213, WO 99/19467, WO 00/60059, and WO 02/10355 (all documents hereby incorporated by reference). Specifically contemplated alpha-amylase variants are disclosed in U.S. Pat. Nos. 6,093,562, 6,297,038 or U.S. Pat. No. 6,187,576 (hereby incorporated by reference) and include Bacillus stearothermophilus alpha-amylase (BSG alpha-amylase) variants having a deletion of one or two amino acid in positions R179 to G182, preferably a double deletion disclosed in WO 96/23873—see, e.g., page 20, lines 1-10 (hereby incorporated by reference), preferably corresponding to delta(181-182) compared to the wild-type BSG alpha-amylase amino acid sequence set forth in SEQ ID NO:3 disclosed in WO 99/19467 or deletion of amino acids R179 and G180 using SEQ ID NO:3 in WO 99/19467 for numbering (which reference is hereby incorporated by reference). Even more preferred are Bacillus alpha-amylases, especially Bacillus stearothermophilus alpha-amylase, which have a double deletion corresponding to delta(181-182) and further comprise a N193F substitution (also denoted 1181*+G182*+N193F) compared to the wild-type BSG alpha-amylase amino acid sequence set forth in SEQ ID NO:3 disclosed in WO 99/19467.

Bacterial Hybrid Alpha-Amylase

A hybrid alpha-amylase specifically contemplated comprises 445 C-terminal amino acid residues of the Bacillus licheniformis alpha-amylase (shown in SEQ ID NO: 4 of WO 99/19467) and the 37 N-terminal amino acid residues of the alpha-amylase derived from Bacillus amyloliguefaciens (shown in SEQ ID NO: 5 of WO 99/19467), with one or more, especially all, of the following substitution:

-   G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S (using the     Bacillus licheniformis numbering in SEQ ID NO: 4 of WO 99/19467).     Also preferred are variants having one or more of the following     mutations (or corresponding mutations in other Bacillus     alpha-amylase backbones): H154Y, A181T, N190F, A209V and Q264S     and/or deletion of two residues between positions 176 and 179,     preferably deletion of E178 and G179 (using the SEQ ID NO: 5     numbering of WO 99/19467). In an embodiment the bacterial     alpha-amylase is dosed in an amount of 0.0005-5 KNU per g DS,     preferably 0.001-1 KNU per g DS, such as around 0.050 KNU per g DS.

Fungal Alpha-Amylase

Fungal Alpha-Amylases Include Alpha-Amylases Derived from a Strain of the Genus Aspergillus, such as Aspergillus oryzae, Aspergillus niger and Aspergillis kawachii Alpha-Amylases.

A preferred acidic fungal alpha-amylase is a Fungamyl-like alpha-amylase which is derived from a strain of Aspergillus oryzae. According to the present invention, the term “Fungamyl-like alpha-amylase” indicates an alpha-amylase which exhibits a high identity, i.e., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity to the mature part of the amino acid sequence shown in SEQ ID NO: 10 in WO 96/23874. Another preferred acid alpha-amylase is derived from a strain Aspergillus niger. In a preferred embodiment the acid fungal alpha-amylase is the one from Aspergillus niger disclosed as “AMYA_ASPNG” in the Swiss-prot/TeEMBL database under the primary accession no. P56271 and described in WO 89/01969 (Example 3—incorporated by reference). A commercially available acid fungal alpha-amylase derived from Aspergillus niger is SP288 (available from Novozymes NS, Denmark). Other contemplated wild-type alpha-amylases include those derived from a strain of the genera Rhizomucor and Meripilus, preferably a strain of Rhizomucor pusillus (WO 2004/055178 incorporated by reference) or Meripilus giganteus. In a preferred embodiment the alpha-amylase is derived from Aspergillus kawachii and disclosed by Kaneko et al., 1996, J. Ferment. Bioeng. 81: 292-298, “Molecular-cloning and determination of the nucleotide-sequence of a gene encoding an acid-stable alpha-amylase from Aspergillus kawachii, and further as EMBL: #AB008370. The fungal alpha-amylase may also be a wild-type enzyme comprising a starch-binding domain (SBD) and an alpha-amylase catalytic domain (i.e., non-hybrid), or a variant thereof. In an embodiment the wild-type alpha-amylase is derived from a strain of Aspergillus kawachii.

Fungal Hybrid Alpha-Amylase

In a preferred embodiment the fungal acid alpha-amylase is a hybrid alpha-amylase. Preferred examples of fungal hybrid alpha-amylases include the ones disclosed in WO 2005/003311 or U.S. Patent Publication no. 2005/0054071 (Novozymes) or U.S. patent application No. 60/638,614 (Novozymes) which is hereby incorporated by reference. A hybrid alpha-amylase may comprise an alpha-amylase catalytic domain (CD) and a carbohydrate-binding domain/module (CBM), such as a starch binding domain, and optional a linker. Specific examples of contemplated hybrid alpha-amylases include those disclosed in Table 1 to 5 of the examples in U.S. patent application No. 60/638,614, including Fungamyl variant with catalytic domain JA118 and Athelia rolfsii SBD (SEQ ID NO:100 in U.S. 60/638,614), Rhizomucor pusillus alpha-amylase with Athelia rolfsii AMG linker and SBD (SEQ ID NO:101 in U.S. 60/638,614), Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and SBD (which is disclosed in Table 5 as a combination of amino acid sequences SEQ ID NO:20, SEQ ID NO:72 and SEQ ID NO:96 in U.S. application Ser. No. 11/316,535) or as V039 in Table 5 in WO 2006/069290, and Meripilus giganteus alpha-amylase with Athelia rolfsii glucoamylase linker and SBD (SEQ ID NO:102 in U.S. 60/638,614). Other specifically contemplated hybrid alpha-amylases are any of the ones listed in Tables 3, 4, 5, and 6 in Example 4 in U.S. application Ser. No. 11/316,535 and WO 2006/069290 (hereby incorporated by reference). Other specific examples of contemplated hybrid alpha-amylases include those disclosed in U.S. Patent Publication no. 2005/0054071, including those disclosed in Table 3 on page 15, such as Aspergillus niger alpha-amylase with Aspergillus kawachii linker and starch binding domain. Contemplated are also alpha-amylases which exhibit a high identity to any of above mention alpha-amylases, i.e., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity to the mature enzyme sequences. An acid alpha-amylases may according to the invention be added in an amount of 0.001 to 10 AFAU/g DS, preferably from 0.01 to 5 AFAU/g DS, especially 0.3 to 2 AFAU/g DS or 0.001 to 1 FAU-F/g DS, preferably 0.01 to 1 FAU-F/g DS.

Commercial Alpha-Amylase Products

Preferred commercial compositions comprising alpha-amylase include MYCOLASE™ from DSM (Gist Brocades), BAN™, TERMAMYL™ SC, FUNGAMYL™, LIQUOZYME™ X, LIQUOZYME™ SC and SAN™ SUPER, SAN™ EXTRA L (Novozymes NS) and CLARASE™ L-40,000, DEX-LO™, SPEZYME™ FRED, SPEZYME™ AA, and SPEZYME™ DELTA AA, SPEZYME™ XTRA, GC358 (Genencor Int.), FUELZYME™-LF (Verenium Inc), and the acid fungal alpha-amylase sold under the trade name SP288 (available from Novozymes NS, Denmark).

Carbohydrate-Source Generating Enzyme

The term “carbohydrate-source generating enzyme” includes glucoamylase (being glucose generators), beta-amylase and maltogenic amylase (being maltose generators) and also alpha-glucosidase. A carbohydrate-source generating enzyme is capable of producing a carbohydrate that can be used as an energy-source by the fermenting organism(s) in question, for instance, when used in a process of the invention for producing a fermentation product, such as ethanol. The generated carbohydrate may be converted directly or indirectly to the desired fermentation product, preferably ethanol. According to the invention a mixture of carbohydrate-source generating enzymes may be used. Especially contemplated blends are mixtures comprising at least a glucoamylase and an alpha-amylase, especially an acid amylase, even more preferred an acid fungal alpha-amylase. The ratio between glucoamylase activity (AGU) and fungal alpha-amylase activity (FAU-F) (i.e., AGU per FAU-F) may in a preferred embodiment of the invention be between 0.1 and 100 AGU/FAU-F, in particular between 2 and 50 AGU/FAU-F, such as in the range from 10-40 AGU/FAU-F, especially when doing one-step fermentation (Raw Starch Hydrolysis—RSH), i.e., when saccharification and fermentation are carried out simultaneously (i.e., without a liquefaction step). In a conventional starch-to-ethanol process (i.e., including a liquefaction step (a)) the ratio may preferably be as defined in EP 140,410-B1, especially when saccharification in step (b) and fermentation in step (c) are carried out simultaneously.

Glucoamylase

A glucoamylase used according to the invention may be derived from any suitable source, e.g., derived from a microorganism or a plant. Preferred glucoamylases are of fungal or bacterial origin, selected from the group consisting of Aspergillus glucoamylases, in particular Aspergillus niger G1 or G2 glucoamylase (Boel et al., 1984, EMBO J. 3(5): 1097-1102), or variants thereof, such as those disclosed in WO 92/00381, WO 00/04136 and WO 01/04273 (from Novozymes, Denmark); the A. awamori glucoamylase disclosed in WO 84/02921, Aspergillus oryzae glucoamylase (Agric. Biol. Chem. 55(4): 941-949 (1991)), or variants or fragments thereof. Other Aspergillus glucoamylase variants include variants with enhanced thermal stability: G137A and G139A (Chen et al., 1996, Prot. Eng. 9: 499-505); D257E and D293E/Q (Chen et al., 1995, Prot. Eng. 8: 575-582); N182 (Chen et al., 1994, Biochem. J. 301: 275-281); disulphide bonds, A246C (Fierobe et al., 1996, Biochemistry 35: 8698-8704; and introduction of Pro residues in position A435 and S436 (Li et al., 1997, Protein Eng. 10: 1199-1204. Other glucoamylases include Athelia rolfsii (previously denoted Corticium rolfsii) glucoamylase (see U.S. Pat. No. 4,727,026 and (Nagasaka et al., 1998, “Purification and properties of the raw-starch-degrading glucoamylases from Corticium rolfsii, Appl Microbiol Biotechnol. 50:323-330), Talaromyces glucoamylases, in particular derived from Talaromyces emersonii (WO 99/28448), Talaromyces leycettanus (U.S. Pat. No. Re. 32,153), Talaromyces duponti, Talaromyces thermophilus (U.S. Pat. No. 4,587,215). Bacterial glucoamylases contemplated include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum (EP 135,138), and C. thermohydrosulfuricum (WO 86/01831) and Trametes cingulata, Pachykytospora papyracea; and Leucopaxillus giganteus all disclosed in WO 2006/069289; or Peniophora rufomarginata disclosed in WO 2007/124285; or a mixture thereof. Also hybrid glucoamylase are contemplated according to the invention. Examples the hybrid glucoamylases disclosed in WO 2005/045018. Specific examples include the hybrid glucoamylase disclosed in Table 1 and 4 of Example 1 (which hybrids are hereby incorporated by reference). Contemplated are also glucoamylases which exhibit a high identity to any of above mention glucoamylases, i.e., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity to the mature enzymes sequences mentioned above. Commercially available compositions comprising glucoamylase include AMG 200L; AMG 300 L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™ FUEL, SPIRIZYME™ B4U, SPIRIZYME™ ULTRA and AMG™ E (from Novozymes A/S); OPTIDEX™ 300, GC480, GC417 (from Genencor Int.); AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™ G900, G-ZYME™ and G990 ZR (from Genencor Int.). Glucoamylases may in an embodiment be added in an amount of 0.0001-20 AGU/g DS, preferably 0.001-10 AGU/g DS, especially between 0.01-5 AGU/g DS, such as 0.1-2 AGU/g DS.

Beta-Amylase

A beta-amylase (E.C3.2.1.2) is the name traditionally given to exo-acting maltogenic amylases, which catalyze the hydrolysis of 1,4-alpha-glucosidic linkages in amylose, amylopectin and related glucose polymers. Maltose units are successively removed from the non-reducing chain ends in a step-wise manner until the molecule is degraded or, in the case of amylopectin, until a branch point is reached. The maltose released has the beta anomeric configuration, hence the name beta-amylase. Beta-amylases have been isolated from various plants and microorganisms (Fogarty et al., 1979, Progress in Industrial Microbiology 15: 112-115). These beta-amylases are characterized by having optimum temperatures in the range from 40° C. to 65° C. and optimum pH in the range from 4.5 to 7. A commercially available beta-amylase from barley is NOVOZYM™ WBA from Novozymes A/S, Denmark and SPEZYME™ BBA 1500 from Genencor Int., USA.

Maltogenic Amylase

The amylase may also be a maltogenic alpha-amylase. A “maltogenic alpha-amylase” (glucan 1,4-alpha-maltohydrolase, E.C. 3.2.1.133) is able to hydrolyze amylose and amylopectin to maltose in the alpha-configuration. A maltogenic amylase from Bacillus stearothermophilus strain NCIB 11837 is commercially available from Novozymes A/S. Maltogenic alpha-amylases are described in U.S. Pat. Nos. 4,598,048, 4,604,355 and 6,162,628, which are hereby incorporated by reference. The maltogenic amylase may in a preferred embodiment be added in an amount of 0.05-5 mg total protein/gram DS or 0.05-5 MANU/g DS.

Materials & Methods Materials:

-   Parent Pullulanase H: Thermococcus hydrothermalis pullulanase     (Uniprot: Q9Y8I8) disclosed in SEQ ID NO: 2. -   Parent Pullulanase L: Thermococcus litoralis pullulanase (UNIPROT:     Q8NKS8 and disclosed in SEQ ID NO: 4. -   Alpha-Amylase FZ: Alpha-amylase as disclosed in Richardson et al.,     2002, The Journal of Biological Chemistry 277(29): 267501-26507     (Issue 19 July), referred to as BD5088. This alpha-amylase is the     same as the one shown in SEQ ID NO: 4 herein. The mature enzyme     sequence starts after the initial “Met” amino acid in position 1.     The enzyme is available from Verenium (USA). -   Alpha-Amylase SC: Bacillus stearothermophilus alpha-amylase, which     has a double deletion corresponding to delta(181-182) and further     comprise a N193F substitution. -   Protease A: Metallo protease derived from Thermoascus aurantiacus     CGMCC No. 0670 disclosed as amino acids 1-177 in SEQ ID NO: 1 herein     and amino acids 1-177 in SEQ ID NO: 2 in WO 2003/048353.

Glucoamylase SF: Blend of Tamaromyces emersonii glucoamylase disclosed in WO 99/28448 as SEQ ID NO: 7 and Trametes cingulata glucoamylase disclosed in WO06/069289 in a ratio of about 9:1.

-   BMSY medium: (1% yeast extract, 2% peptone, 100 mM potassium     phosphate buffer, pH 6.0, 1.34% YNB, 4×10-5% biotin, 2% sorbitol)

Methods: Hidden Markov Model (HMM):

The strategy for creating the Hidden Markov Model is as indicated below. The model is constructed using a multiple alignment of X47 domain sequences shown in SEQ ID NO: 20-30 as the only input:

-   1) Extract sequences which are used for the model (Cazy/eFAM) -   2) Make a multiple alignment of those sequences -   3) Using ‘hmmbuild’ (part of hmmer package v. 2.3.2) for     constructing the model -   4) The model is calibrated using random sequences, using     hmmcalibrate -   5) The program ‘hmmsearch’ can then be used to query any database     using the model.

Tasks 2-4 are repeated on all sequences that identified using the first model. This provides more diversity to the model, going from 5 to 11 sequences included.

Alpha-Amylase Activity (KNU)

The amylolytic activity may be determined using potato starch as substrate. This method is based on the break-down of modified potato starch by the enzyme, and the reaction is followed by mixing samples of the starch/enzyme solution with an iodine solution. Initially, a blackish-blue color is formed, but during the break-down of the starch the blue color gets weaker and gradually turns into a reddish-brown, which is compared to a colored glass standard.

One Kilo Novo alpha amylase Unit (KNU) is defined as the amount of enzyme which, under standard conditions (i.e., at 37° C.+/−0.05; 0.0003 M Ca²⁺; and pH 5.6) dextrinizes 5260 mg starch dry substance Merck Amylum solubile. A folder EB-SM-0009.02/01 describing this analytical method in more detail is available upon request to Novozymes NS, Denmark, which folder is hereby included by reference.

Determination of FAU Activity

One Fungal Alpha-Amylase Unit (FAU) is defined as the amount of enzyme, which breaks down 5.26 g starch (Merck Amylum solubile Erg. B.6, Batch 9947275) per hour based upon the following standard conditions:

Substrate Soluble starch Temperature 37° C. pH 4.7 Reaction time 7-20 minutes

Determination of Acid Alpha-Amylase Activity (AFAU)

Acid alpha-amylase activity is measured in AFAU (Acid Fungal Alpha-amylase Units), which are determined relative to an enzyme standard.

The standard used is AMG 300 L (from Novozymes A/S, Denmark, glucoamylase wild-type Aspergillus niger G1, also disclosed in Boel et al., 1984, EMBO J. 3(5): 1097-1102) and WO 92/00381). The neutral alpha-amylase in this AMG falls after storage at room temperature for 3 weeks from approx. 1 FAU/mL to below 0.05 FAU/mL.

The acid alpha-amylase activity in this AMG standard is determined in accordance with the following description. In this method, 1 AFAU is defined as the amount of enzyme, which degrades 5.260 mg starch dry matter per hour under standard conditions Iodine forms a blue complex with starch but not with its degradation products. The intensity of color is therefore directly proportional to the concentration of starch. Amylase activity is determined using reverse colorimetry as a reduction in the concentration of starch under specified analytic conditions.

Alpha-amylase Starch + Iodine → Dextrins + Oligosaccharides 40° C., pH 2.5 Blue/violet t = 23 sec. Decoloration

-   -   Standard conditions/reaction conditions: (per minute)     -   Substrate: Starch, approx. 0.17 g/L     -   Buffer: Citate, approx. 0.03 M     -   Iodine (I₂): 0.03 g/L     -   CaCl₂: 1.85 mM     -   pH: 2.50±0.05     -   Incubation temperature: 40° C.     -   Reaction time: 23 seconds     -   Wavelength: lambda=590 nm     -   Enzyme concentration: 0.025 AFAU/mL     -   Enzyme working range: 0.01-0.04 AFAU/mL

If further details are preferred these can be found in EB-SM-0259.02/01 available on request from Novozymes A/S, Denmark, and incorporated by reference.

Glucoamylase Activity (AGU)

The Novo Glucoamylase Unit (AGU) is defined as the amount of enzyme, which hydrolyzes 1 micromole maltose per minute under the standard conditions 37° C., pH 4.3, substrate: maltose 23.2 mM, buffer: acetate 0.1 M, reaction time 5 minutes. An autoanalyzer system may be used. Mutarotase is added to the glucose dehydrogenase reagent so that any alpha-D-glucose present is turned into beta-D-glucose. Glucose dehydrogenase reacts specifically with beta-D-glucose in the reaction mentioned above, forming NADH which is determined using a photometer at 340 nm as a measure of the original glucose concentration.

AMG incubation: Substrate: maltose 23.2 mM Buffer: acetate 0.1M pH: 4.30 ± 0.05 Incubation temperature: 37° C. ± 1 Reaction time: 5 minutes Enzyme working range: 0.5-4.0 AGU/mL Color reaction: GlucDH: 430 U/L Mutarotase: 9 U/L NAD: 0.21 mM Buffer: phosphate 0.12M; 0.15M NaCl pH: 7.60 ± 0.05 Incubation temperature: 37° C. ± 1 Reaction time: 5 minutes Wavelength: 340 nm

A folder (EB-SM-0131.02/01) describing this analytical method in more detail is available on request from Novozymes A/S, Denmark, which folder is hereby included by reference.

Protease Assays

The following assays for protease activity were used:

AZCL-Casein Assay

A solution of 0.2% of the blue substrate AZCL-casein is suspended in Borax/NaH₂PO₄ buffer pH9 while stirring. (For pH profile the buffer system pH 3 to pH 11 is used instead). The solution is distributed while stirring to microtiter plate (100 microL to each well), 30 microL enzyme sample is added and the plates are incubated in an Eppendorf Thermomixer for 30 minutes at 45° C. and 600 rpm. Denatured enzyme sample (100° C. boiling for 20 min) is used as a blank. After incubation the reaction is stopped by transferring the microtiter plate onto ice and the coloured solution is separated from the solid by centrifugation at 3000 rpm for 5 minutes at 4° C. 60 microL of supernatant is transferred to a microtiter plate and the absorbance at 595nm is measured using a BioRad Microplate Reader.

pNA-Assay

50 microL protease sample is added to a microtiter plate and the assay is started by adding 100 microL 1 mM pNA substrate (5 mg dissolved in 100 microL DMSO and further diluted to 10 mL with Borax/NaH₂PO₄ buffer pH9.0). The increase in OD₄₀₅ at room temperature is monitored as a measure of the protease activity.

Determination of Maltogenic Amylase activity (MANU)

One MANU (Maltogenic Amylase Novo Unit) may be defined as the amount of enzyme required to release one micro mole of maltose per minute at a concentration of 10 mg of maltotriose (Sigma M 8378) substrate per ml of 0.1 M citrate buffer, pH 5.0 at 37° C. for 30 minutes.

Protein Concentration Determination

The protein concentration of the enzyme preparation is determined initially by measuring the absorbance of triplicate samples at 280 nm and then calculating the protein concentration using Lambert-Beers law and a conversion factor (E) of 2.20. The conversion factor is based on the theoretical molar extinction coefficient which in turn is obtained from the sequence. In addition to the absorbance measurement, samples are sent to amino acid analysis by acid hydrolysis to obtain a more accurate measure of the enzyme concentration. In the time between sampling and receiving the data from the amino acid analysis the enzyme concentration estimate from the absorbance measurement is used for subsequent experiments.

Determination of pH optimum of Pullulanases

The pH optima for the enzymes are measured at 6 pHs (3, 4, 5, 6, 7 and 8) and at different dilutions of protein as required. All data points are results of duplicate measurements. Substrate solutions are prepared containing 0.2% AZCL-pullulan in Britton Robinson buffer (50 mM phosphate, 50 mM Succinate, 50 mM Borate) which provides buffering capacity in the interval between pH 2 and 11. 270 microliters substrate solution is then transferred to a 96-well micro-titer plate and 18 microliters enzyme solution added. After addition of enzyme, the plates are incubated at 40° C. in a thermo-block with shaking for 15 minutes and then centrifuged using a centrifuge rotor specially adapted for micro-titer plates. After centrifugation 150 microliters of the supernatant is transferred to a new micro-titer plate and the absorbance at 595 nm measured.

Determination of Temperature Optimum of Pullulanases

The temperature optima for the enzymes are measured using a slightly modified version of the AZCL-pullulan protocol. Triplicate samples containing 750 microliters substrate solution (0.2% AZCL pullulan in 25 mM Na Acetate, pH 4.5) are prepared for each temperature. The samples are then pre-warmed to the desired temperatures for 5 minutes. After warming 50 microliters enzyme solution is added. The tubes are then directly put in thermo-blocks set at the desired temperatures and incubated with shaking for 20 minutes. After incubation the samples are put on ice for 2 minutes and then centrifuged in a bench-top centrifuge to pellet the un-degraded substrate. A volume of the supernatant is then withdrawn from each sample and added to a well in a 96-well micro-titer plate and diluted with substrate solution without AZCL-pullulan as necessary. The absorbance of each sample is then measured at 650 nm and the standard deviation of the triplicate samples calculated for each temperature.

Determination of Inactivation Temperature

Inactivation of pullulanase is tested by incubating a 1 ml 0.29 mg/ml sample at 85° C. for 1.5 hours. After incubation the sample is put on ice for 5 minutes to cool down and then centrifuged at maximum speed at 4° C. in a bench top centrifuge for 5 minutes. 600 microliters of the supernatant is then carefully removed and transferred to a new tube. 5×50 microliters supernatant is then added to five tubes containing 750 microliters AZCL-pullulan in 50 mM Na Acetate, pH 4.5. The samples are then incubated at 60° C. for 20 minutes in a thermo-block with constant shaking. In parallel to the heat inactivated sample a non-heat incubated sample is treated and assayed exactly like the heat treated sample to serve as a negative control and for normalization of the data.

Determination of Temperature Stability (DSC)

The temperature stability of the enzyme is determined by differential scanning calorimetry (DSC). Briefly, a 2.5 ml 1 mg/ml enzyme sample is prepared by diluting 431 microliters of the T. litoralis—T. hydrothermalis pullulanase chimera with DSC buffer (10 mM NaAc, 50 mM NaCl, pH 5.0) to the desired volume. The enzyme sample is then applied to a PD10 desalting column pre equilibrated with 25 of the DSC buffer and eluted with an additional 3.5 mL of the same buffer. The enzyme sample and a reference sample consisting of the DSC buffer without enzyme is then degassed for 45 minutes to remove dissolved air which could interfere with the experiment. After degassing, 512 microliters of the enzyme solution and of the DSC buffer are loaded into the sample cell and the reference cell respectively. Scanning is then done once between 20° and 120° with a heating rate of 90° per hour. The maximum of the resulting peak in the thermo gram is taken to be the denaturation temperature or Td.

EXAMPLES Example 1

Cloning and Expression of Thermococcus hydrothermalis DSM 14834 Pullulanase in Bacillus subtilis

A synthetic gene based on the protein sequence of Thermococcus hydrothermalis DSM 14834 Apu (Uniprot: Q9Y8I8) was designed and the gene was codon optimized for Bacillus subtilis. The C-terminal sequence (including the part of the putative linker, indicated in the alignment—FIG. 1) TPTESPTETTTTTPSETTTTTSTTTGPSSTTTSTPGGGICGPGIIAGLALIPLLLKRRN (SEQ ID NO: 5) and the native signal peptide (MRRVVALFIAILMLGSIVGANVKSVG—SEQ ID NO: 6) was not part of the designed synthetic gene (SEQ ID NO: 1 (DNA) and SEQ ID NO: 2 (protein)).The synthetic gene was extracted by double-digestion according to the manufacturer's manual (FastDigest®, Fermentas, Germany) from a plasmid carrying the synthetic gene. The synthetic gene was cloned in an expression vector comprising the genetic elements as described in WO 99/43835 (hereby incorporated by reference). By doing so, the signal peptide from the alkaline protease from Bacillus clausii (aprH) was fused to the synthetic gene as described in WO 99/43835 in frame to the DNA encoding the truncated apu gene. Alternatively, the aprH signal peptide was extended N-terminally by the amino acids HQHQHQHPR which enabled us to purify the mature peptide by affinity chromatography.

aprH signal peptide: (SEQ ID NO: 18) MKKPLGKIVASTALLISVAFSSSIASA aprH signal peptide including HQ tag: (SEQ ID NO: 19) MKKPLGKIVASTALLISVAFSSSIASAHQHQHQHPR

The derived expression plasmidC1MW containing the apu coding sequence with aprH signal plus HQ affinity tag was integrated by homologous recombination into a Bacillus subtilis host cell genome. The gene construct was expressed under the control of a triple promoter system (as described in WO 99/43835). The gene coding for chloramphenicol acetyltransferase was used as maker (as described in (Diderichsen et al., 1993, Plasmid 30: 312-315). This cloning work resulted in truncation site X1 of T. hydrothermalis pullulanase (FIG. 2).

Chloramphenicol resistant clones were analyzed by DNA sequencing to verify the correct DNA sequence of the construct. One expression clone was selected and was cultivated on a rotary shaking table in 500 ml baffled Erlenmeyer flasks each containing 100 ml casein based media supplemented with 6 mg/l chloramphenicol. The clone was cultivated for 3-5 days at 37° C. Pullulanase activity in the culture broth was determined as described in Example 4.

Example 2 Cloning and Expression of Variants of Thermococcus hydrothermalis DSM 14834 Pullulanase in Bacillus subtilis

In order to increase the recombinant expression yield of Apu and to identify which domains are necessary for the pullulanase activity, we have made truncations of the enzyme. The expression plasmid containing the synthetic gene described in Example 1 was used as template for a PCR(1). PCR(1) was performed in a total volume of 26 microliters, the following reagents were added, 1 microliter of vector DNA preparation (template), 50 pmol of each of the primer pairs below, 5 microliters dNTPs and 0.5 microliter Phusion® polymerase (Finnzymes, Finland) in Phusion HF buffer. The PCR conditions were 98° C. for 2 min; 9 cycles of 98° C. for 15 sec; 65° C. for 45 sec; 72° C. for 4 min; followed by 72° C. for 10 min; 4° C. for 20 min and 15° C. until the end of the PCR program. The primer pairs used were:

For X4 truncation:

For X4 truncation: 1354 (SEQ ID NO: 31) 5′- GCCAAGGCCGGTTTTTTATGTTTTACTTAAGGATTACGCGAGCAT TG and C52X4r (SEQ ID NO: 9) 5′- TGATTAACGCGTTTAAGTATAGTTGCCAGGGCCATGG For X5 truncation: 1354 (SEQ ID NO: 31) 5′- GCCAAGGCCGGTTTTTTATGTTTTACTTAAGGATTACGCGAGCAT TG and C52X5r (SEQ ID NO: 10) 5′- TGATTAACGCGTTTAAGGAGGCTCAACGC

The obtained PCR products were each cloned into an expression vector as described in Example 1. The DNA sequences of the derived expression vectors were verified by Sanger sequencing which is within the general knowledge of one skilled in the art.

The plasmids were each integrated by homologous recombination into the Bacillus subtilis host cell genome leading to truncations X4 and X5. Genomic DNA from each of the cultivated clones was prepared and used to transform a protease weak Bacillus subtilis host strain. Selection of one clone and pullulanase activity determination was performed as described in Example 1.

Example 3

Cloning and Expression of a Truncated Pullulanase Fusion of Thermococcus litoralis DSM 5473 and Thermococcus hydrothermalis DSM 14834 in Bacillus subtilis

A truncated synthetic gene based on the protein sequence of Thermococcus litoralis DSM 5473 was designed and the gene was codon optimized for Bacillus subtilis. In the synthetic gene, the C-terminal sequence after amino acid 474 in SEQ ID NO: 4 was deleted as indicated in the alignment FIG. 2 (“X6 truncation”).

Cloning of the synthetic gene into Bacillis subtilis the selection of correct clones and the cultivation of the clones was performed as described in Example 1. The X6 truncation of T. litoralis did not display any pullulanase activity (see table in Example 4). The derived expression plasmid C526A was used in a following PCR amplification as template.

Fusion of Thermococcus litoralis and Thermococcus hydrothermalis DNA

Using the expression plasmid C526A carrying the apu gene from Thermococcus litoralis as template, a PCR was performed in a total volume of 50 microliters. The following reagents were added, 1 microL of the template DNA, 50 pmol of each of the primers (SEQ ID NOS: 19 and 8), dNTPs and Phusion® polymerase (Finnzymes, Finland) in Phusion HF buffer. The PCR conditions were 98° C. for 30 sec; 36 cycles of 98° C. for 10 sec; 65° C. for 20 sec; 72° C. for 90 sec; followed by 72° C. for 10 min; and 4° C. until the end of the PCR program.

The primers used were:

-   Tlito.r 5′-CGGCGTAAGCTTGTTTGCCT (SEQ ID NO: 7) 1354     5′-GCCAAGGCCGGTTTTTTATGTTTTACTTAAGGATTACGCGAGCATTG (SEQ ID NO: 31)     where SEQ ID NO: 31 binds in a genetic element of Bacillus subtilis     as referenced in Example 1 and SEQ ID NO: 7 binds on the reverse     strand of the apu synthetic gene from Thermococcus litoralis.

The expression plasmid containing the Thermococcus hydrothermalis synthetic gene described in Example 1 was used as template for another PCR reaction which was performed under the same conditions as described above with the exception that the primers used were:

C52X4r (SEQ ID NO: 32) 5′-TGATTAACGCGTTTAAGTATAGTTGCCAGGGCCATGG Fusion.f (SEQ ID NO: 8) 5′-aggcaaacaagcttacgccgcgcatgatggagcgcctt

The obtained PCR products from both PCR reactions from Example 3 were purified under recommended conditions from the manufacturer (GFX DNA purification kit, GE Healthcare). The purified PCR products where used as template in a fusion PCR which was performed in a total volume of 50 microliters, the following reagents were added, 1 microliter of a 5 times dilution of each PCR fragments in water, 50 pmol of each of the primers SEQ ID NOs: 31 and 32, dNTPs and Phusion® polymerase (Finnzymes, Finland) in Phusion HF buffer. The PCR conditions were 98° C. for 30 sec; 36 cycles of 98° C. for 10 sec; 65° C. for 20 sec; 72° C. for 150 sec; followed by 72° C. for 10 min; and 4° C. until the end of the PCR program.

The fusion PCR reaction product of approx. 3 kb was purified and integrated by homologous recombination into the Bacillus subtilis host cell genome leading to a T. hydrothermalis/T. litoralis hybrid enzyme with truncation site X4 (see FIG. 2), i.e., the hybrid has an amino acid sequence from 1-782 in SEQ ID NO: 34. Genomic DNA of one correct clone was prepared and used to transform a protease weak Bacillus subtilis host strain. Selection of one clone and pullulanase activity determination was performed as described in Example 1. Likewise, another hybrid was produced by using primer SEQ ID NO: 33 instead of SEQ ID NO: 32 which yielded in a T. hydrothermalis/T. litoralis hybrid enzyme with truncation site X5 (FIG. 2), i.e., the hybrid has the amino acid sequence from position 1-581 in SEQ ID NO: 34.

Example 4 Determination of Pullulanase Activity

AZCL-pullulan plates with different pH were prepared as follows: 115.2 ml Britton-Robinson buffer and 284.8 ml deionized water were mixed and pH was adjusted to 4.5 or 7.0 by adding 2 N NaOH. Subsequently 4 g Agarose type II (Sigma A6877) was added and the solution was heated with stirring until the agarose was dissolved. After cooling down (to around 55° C.) with stirring, approx. 0.025% AZCL-Pullulan (Megazyme) was added and 70 ml was poured in a 14 cm plate. Wells with 4 mm diameter were punched out.

Amylopectin plates were prepared as follows. 400 ml BT-agar (6.25 g/L Tryptone, 6.25 g/L Amylopectin Hydrate, 25 g/L granulated agar in ‘ionbytte’ water) at 55° C. was mixed with 100 ml Ba2 (1 g/L (NH₄)₂SO₄, 2.5 g/L MgSO₄.7H₂O, 1.25 g/L CaCl₂.2H₂O, 15 g/L KH₂PO₄ in ‘ionbyttet’ water) and 70 ml was poured in a 14 cm plate. 500 microliters samples were taken from the cultures described in the previous examples and centrifuged at 10.000 rpm for 10 min at 12° C. 10 μl of the supernatant was spotted on the different activity plates and incubated at 70° C. or 80° C. After approx. 16 hrs, activity of the different clones was scored by measuring the diameter of the halos (see Table below, numbers are halo diameter in mm, 0 indicates no observed activity).

AZCL AZCL Construct Truncation pullulan pullulan ATBa2 no. Donor site pH 4.5 pH 7.0 pH 5.5 B. subtilis Negative control — 0 0 0 host strain C1MW T. hydrothermalis X1 4 2 3 C52X4 T. hydrothermalis X4 5 7 6 C52X5 T. hydrothermalis X5 0 0 0 C526A T. litoralis X6 0 0 0 C629Z T. litoralis + X4 9 8 3 hydrothermalis fusion site at X6 C629Y T. litoralis + X5 2 0 8 hydrothermalis fusion site at X6

Truncation X4 showed increased activity over a broad pH range compared to the longest X1 truncation construct. The X4 hybrid showed overall greater activity than the Thermococcus hydrothermalis X4 truncation. Truncations X5 and X6 completely lost their pullulanase activity, only minimal activity was observed for the X5 hybrid at pH 4.5. This indicates the importance of the X47 domain for enzyme activity, which is missing in X5 and X6 truncated constructs.

Example 5

Cloning and Expression of Thermococcus hydrothermalis DSM14834 Pullulanase in Pichia pastoris.

A synthetic gene based on the mature form of protein Thermococcus hydrothermalis DSM 14834 Apu (Uniprot: Q9Y8I8) was designed and the gene was codon optimized for Pichia pastoris. The C-terminal sequence (putative linker) EFHQHQHQHQHQHP (SEQ ID NO: 12) and the Signal peptide: (MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLF INTTIASIAAKEEGVSLEKR—SEQ ID NO: 13) was not part of the designed synthetic gene (SEQ ID NO: 14 (DNA) and SEQ ID NO: 15 (mature protein sequence).

The synthetic gene was amplified from a plasmid carrying the synthetic gene by Phusion polymerase with

forward primer: (SEQ ID NO: 16) (AGGGGTATCTCTCGAGAAAAGAGCTGAGCCAAAGCCTTTGAACG and reverse primer: (SEQ ID NO: 17) GGTGCTGATGGAATTCTGGCTCCTCTCCACCAGTTC.

The amplification is under recommended conditions from the manufacturer. The resulting PCR fragment was purified by gel extraction kit, and subcloned into a pichia expression vector pLIZG8HQ at XhoI and EcoRI sites, according to the standard procedure of Infusion kit. By doing so, the synthetic gene was fused in-frame with alpha-factor signal peptide at the 5′-end, as well as HQ tag at the 3′-end. The screening for the plasmid DNA is also within the general knowledge of one skilled in the art.

The resulting expression construct was sequence confirmed and named pC1QB. The plasmid was transformed into Pichia pastoris using standard electroporation protocol (cf. WO 2004/069872-A1). The resulting transformants were grown in BMSY media for 2 days at 28° C. with vigorous shaking. Then cells were induced for 3 days with a daily supplement of 0.5% methanol. The culture supernatant was screened for pullulanase expression using blue substrate AZCL-HE-pullulan (Megazyme) by microtiter plate assay. The absorbance is measured by BioRad Microplate Reader at 595 nm. For checking of purity and determining the molecular weight of purified pullulanase, the culture supernatant was applied to invitrogen SDS-polyacrylamide gel electrophoresis. The transformant both giving the highest OD595 and the strongest band was chosen for further fermentation and subsequent purification of the pullulanase.

Example 6

Characterization of Pullulanase Variants Expressed in Pichia pastoris

Differing from the methods described in the method section, the pullulanases expressed in P. pastoris were purified and characterized as follows:

Purification of Pullulanase

The supernatant of a Pichia pastoris clone corresponding to truncation site X1 was filtered through a 0.45 micro m filter. The solution was applied to a 40 mL Ni-sepharose column (GE healthcare) equilibrated with 20 mM PBS, 0.3 M NaCl, pH 7.0, and the protein was eluted with a linear increase of imidazole concentration. Fractions from the column were analyzed for pullulanase activity and SDS-PAGE.

Temperature Profile

100 microL 0.4% AZCL-HE-pullulan and 150 μl buffer at pH 4.5 were pre-incubated at 50-95° C. in 1.5 mL tubes for 5 mins before adding 10 microL pullulanase sample. The reactions were performed at corresponding temperature for 30 min with shaking at 1000 rpm for 30 mins. The tubes were put on ice after reaction and 100 microliters supernatant was transferred to a microtiter plate. OD595 was read as a measure of pullulanase. For each enzyme the reaction was performed triplicate.

Temperature 50 60 70 80 85 90 95 Relative activity (%) 50.9 42.9 72.1 100 57.4 40.1 62.4

Temperature Stability

10 microL pullulanase samples and 150 microL buffer at pH 4.5 were incubated at 80° C. for 0, 5, 10, 30, 60 and 120 min. After incubation, 100 microL 0.4% AZCL-HE—pullulan was added to the plate. The reactions were performed at 50° C. for 30 min. 100 microliters supernatant was transferred to a micro-titer plate for reading at OD595.

Incubation time (min) 0 5 10 30 60 120 Relative activity (%) 100 196.6 149.2 163.2 185.9 140.8

pH Profile

pH buffers: 100 mM Succinic acid, HEPES, CHES, CAPSO, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100, pH adjusted to 3.5, 4.0, 4.5, 5.0, 6.0 7.0, 8.0, and 9.0 with HCl and NaOH.

5 microL pullulanase sample, 100 microL0.4% AZCL-HE—pullulan (in MilliQ) and 150 microL 50 mM pH buffer (3.5-9) were mixed in a microtitter plate and placed on ice before reaction. The reactions were performed at 70° C., for 30 min. 100 microL supernatant was transferred to a new microtiter plate. OD595 was read as a measure of pullulanase activity. For each enzyme the reaction was performed in triplicate.

pH 3.5 4 4.5 5 6 7 8 9 Relative 14.8 47.5 76.9 100 76.7 67.4 72.9 66.6 activity (%)

pH Stability

10 microL sample and 150 buffer at pH4.5 were incubated for 0, 5, 10, 30, 60 and 120 min at 80° C. 100 MicroL supernatant was transferred to a new microtiter plate and 50 microL 0.4% AZCL-HE-pullulan was added. OD595 was read as a measure of pullulanase activity. For each enzyme the reaction was performed in triplicate.

Incubation time (min) 0 5 10 30 60 120 Relative activity (%) 17.9 69.2 100 57.9 65.6 32.9

Example 7

Purification of Thermococcus litoralis—Thermococcus hydrothermalis Chimeric Pullulanase

The Bacillus subtilis clone culture supernatant was heat treated at 80° C. for 15 min and then adjusted in pH to 8.0 by addition of NaOH and subsequently centrifuged at 7000 rpm for 20 minutes and filtered through a 0.22 micro-m PES bottle top filter. Purification was done by immobilized metal ion affinity chromatography (IMAC) using an Äkta purifier FPLC system and a Ni-NTA Sepharose FF column. After equilibration of the column with 5 column volumes of Buffer A1 (25 mM Tris/HCl, pH 8.0) the sample was applied. Then the column was washed with 5 column volumes of buffer Al and finally the protein was eluted by a gradient of 0-100% Buffer B1 (25 mM Tris/HCl, 500 mM imidazole, pH 8.0). Fractions were collected throughout the gradient and assayed for pullulanase activity on AZCL-pullulan and protein content by SDS-PAGE. The fractions displaying the highest activity and with protein bands of the correct size in an SDS-PAGE were pooled and buffer shifted by a 1 L G25 desalting column into Buffer A2 (25 mM MES, pH 6.5). The buffer shifted enzyme solution was then applied to a Source 15Q anion exchange column pre-equilibrated with 5 column volumes of Buffer A2. After loading the sample, the column was washed with 5 column volumes of buffer A2 and finally the protein was eluted by a gradient of 0-100% Buffer B2 (25 mM MES, 500 mM NaCl, pH 6.5). Fractions were collected throughout the gradient and assayed for pullulanase activity on AZCL-pullulan and protein content by SDS-PAGE. The fractions with highest activity and purity were pooled and concentrated using Amicon centrifugal filters with a cut off of 30 kDa.

Example 8 Temperature and pH Optimum and Temperature Stability of Pullulanases

The relative temperature optima of the purified pullulanases Thermococcus hydrothermalis (X1 truncation, expressed in Pichia pastoris), the truncation X4 of Thermococcus hydrothermalis and the hybrid (chimer) of Thermococcus hydrothermalis and Thermococcus litoralis (truncation site X4) were determined as described in the method section. FIG. 4 shows the relative activity profiles. The X4 hybrid had the highest temperature optimum of all three pullulanases with 85° C. at pH 4.5.

The temperature stability was measured by DSC determined as described in the Method section. The denaturation temperature for the X4 chimer was 98.5° C. and 86° C. for X4 Thermococcus hydrothermalis.

The pH optimum was determined as described in the Method section. FIG. 5 shows the pH profile of the three pullulanases mentioned above in this example. Truncation X1 and the X4 chimer had the same pH optimum at around pH 5, whereas the truncation X4 of Thermococcus hydrothermalis had a pH optimum that shifted one pH unit higher (pH 6).

Example 9 Liquefaction and Fermentation of Starch-Containing Material

The pullulanase isolated from Thermococcus hydrothermalis and expressed in Pichia pastoris was first application tested in corn slurry liquefaction using a Rapid Visco-Analyzer (RVA). The slurry was made using Corn LP ground corn, backset and tap water. The slurry was made at 32% dry solids (DS), mixed well and then pH adjusted to 5.4 using 50% (w/v) NaOH. The pullulanase was added to the slurry at doses of 1, 5 and 50 micrograms enzyme protein (EP)/g DS from a diluted stock solution made with de-ionized water. Pullulanase was not added to the control mash. Alpha-Amylase SC was used at 0.02% w/w ground corn. The RVA temperature profile was as follows: 5 minute ramp from 65 to 85° C., held at 85° C. for 90 minutes, 10 minute ramp to 32° C. There was continuous mixing at 210 RPM for the entire liquefaction. After liquefaction, the mash weights were adjusted back to the initial weight with tap water to ensure the % dry solids remained approximately the same. The pH of the mashes was adjusted to 5.0 with 40% w/v H₂50₄. Urea and penicillin were added to the mashes at 700 and 3 ppm, respectively. Small scale (4-5 grams mash per fermentation tube) simultaneous saccharification and fermentations (SSF) were run with 5 replicates per mash using Fermentis RedStar® yeast (100 μL inoculum for each replicate; 2.75 g yeast rehydrated in 50 mL tap water for 30 minutes at 32° C.). The Glucoamylase SF dosage (0.5 AGU/g DS) was calculated using the following equation:

${{{Enz}.\mspace{14mu} {dose}}\mspace{14mu} ({ml})} = \frac{\begin{matrix} {{Final}\mspace{14mu} {{enz}.\mspace{14mu} {dose}}\mspace{14mu} \left( {{AGU}\text{/}g\mspace{14mu} {DS}} \right) \times {Mash}\mspace{14mu} {weight}\mspace{14mu} (g) \times} \\ {{Solid}\mspace{14mu} {content}\mspace{14mu} \left( {\% \mspace{14mu} {DS}\text{/}100} \right)} \end{matrix}}{\left( {{{Conc}.\mspace{14mu} {enzyme}}\mspace{14mu} {AGU}\text{/}{ml}} \right)}$

Fermentation progress was measured using CO₂ weight loss over time. The amount of ethanol was calculated using the following equation:

${g\mspace{14mu} {ethanol}\text{/}g\mspace{14mu} {DS}} = \frac{\begin{matrix} {g\mspace{14mu} {CO}_{2}\mspace{14mu} {weight}\mspace{14mu} {loss} \times \frac{1{mol}\mspace{14mu} {CO}_{2}}{44.0098\mspace{14mu} g\mspace{14mu} {CO}_{2}} \times} \\ {\frac{1{mol}\mspace{14mu} {ethanol}}{1{mol}\mspace{14mu} {CO}_{2}} \times \frac{46.094\mspace{14mu} g\mspace{14mu} {ethanol}}{1{mol}\mspace{14mu} {ethanol}}} \end{matrix}}{g\mspace{14mu} {mash}\mspace{14mu} {in}\mspace{14mu} {tube} \times \% \mspace{14mu} {DS}\mspace{14mu} {of}\mspace{14mu} {mash}}$

After 24 hours, one of the replicate tubes for each treatment was sacrificed for HPLC analysis by the addition of 10 microliters/g mash of 40% H₂SO₄, clarified by centrifugation (Beckman Allegra 6R centrifuge with a GH3.8 rotor at 3000 RPM for 10 minutes) and the supernatants filtered through Whatman 0.45 μm syringe filters. The 24 hours samples were diluted 1:5 (v/v) with 5 mM H₂SO₄ buffer prior to HPLC analysis. After 54 hours of fermentation, the remaining 4 tubes for each treatment were stopped as just described. HPLC analysis of ethanol, dextrins and organic acids was on an Agilent 1100/1200 series running a BioRad ion exclusion column with 5 mM H₂SO₄ as the mobile phase and the analytes were detected by a refractive index detector. Analytes were quantified using commercially available standards.

Example 10 Liquefaction and Fermentation of Starch Material using a Pullulanase

The pullulanase from Thermococcus hydrothermalis truncation X2 was added to starch liquefaction at different concentrations as described in Example 9. The 0.5, 1, 2 and 20 micro g EP/g DS doses produced statistically significantly more ethanol than the control (see FIG. 6) using the Student's t-test at the p<0.01 confidence interval. The 5, 10 and 40 micro g EP/g DS doses produced statistically more ethanol that the control at the p<0.05 confidence interval.

Example 11

Identifying X47 Domains using Hidden Markov Model (HMM)

The HMM model was made as described in the “Materials & Method”-section with 11 known family GH57 pullulanase sequences (see point 2) having X47 domains corresponding to positions 580-768 in SEQ ID NO: 2 (pullulanase from Thermococcus hydrothermalis). The program ‘hmmsearch’ was used with the derived HMM model to search the Uniprot database. The individual scores of the pullulanases having X47 domains are shown here:

Organism Accession number HMMscore Thermococcus hydrothermalis SWISSPROT: Q9Y8I8 427.4 Thermococcus sp. HJ21. SWISSPROT: B6SED6 439.3 Thermococcus kodakaraensis SWISSPROT: Q5JJ55 416.9 Thermococcus sp. AM4. SWISSPROT: B7QZQ4 418.1 Pyrococcus furiosus. SWISSPROT: Q8TZQ1 422.2 Pyrococcus furiosus DSM 3638. SWISSPROT: Q3HUR3 422.2 Pyrococcus furiosus. SWISSPROT: O30772 421.7 Thermococcus gammatolerans SWISSPROT: C5A4E3 416.2 DSM 15229 Thermococcus barophilus MP. SWISSPROT: B5IRL5 410.5 Thermococcus litoralis. SWISSPROT: Q8NKS8 414.2 Pyrococcus abyssi. SWISSPROT: Q9V294 394.7

Example 12

Cloning and Expression of Isolated X47 Domain from Amylpullulanase of Thermococcus hydrothermalis DSM 14834

Oligo name Oligo seq C1QBX46-F AGGGGTATCTCTCGAGAAAAGACCATCCTACTT (SEQ ID NO: 35) GTTCGGAAAC C1QBX46-R GGTGCTGATGGAATTCGATGTCGGCGATAACAAC (SEQ ID NO: 36) ACC

The synthetic gene shown in SEQ ID NO: 14 herein was used as template to amplify the isolated domain of the amylopullulanase from T. hydrothermalis. The X47 domain was amplified with Phusion polymerase using oligo pairs mentioned in above table and cloned in Pichia pastoris as described in Example 5.

The sequences of the resulting expression constructs (the coding region of the expressed X47 domain and alpha signal peptide is shown in SEQ ID NO: 37 (DNA) and SEQ ID NO: 38 (Peptide) were confirmed by Sanger sequencing and named pX47. The plasmids were transformed into Pichia pastoris using a standard electroporation protocol (as described in Example 5). The complete CDS of the X47 domain was expressed in Pichia pastoris using the same method as also described in Example 5.”

Purification

The pH of the P. pastoris culture expressing the X47 domain was adjusted to 7.0 with NaOH then filtered through a 0.45 micro-m filter. The solution was applied to a 30 mL Ni-sepharose High Performance column (GE Healthcare) which was equilibrated with 20 mM Tris-HCl containing 0.3 M NaCl at pH 7.0. The protein was eluted with a linear imidazole gradient (0-500 mM). Fractions from the column were analyzed by SDS-PAGE.

Characterization of X47 Domain Amylopullulanase and Isolated X47 Domain Ratio Profile

10 micro g pullulanase from Thermococcus hydrothermalis (P6VK), different amount of X47, 100 microL 0.4% AZCL-HE-pullulan (Megazyme International Ireland Ltd.) and 150 microL buffer at pH 4.5, and different amount of milliQ water (to give the same final volume for different ratio) were mixed and incubated at corresponding temperature for 50 min with shaking at 900 rpm. The tubes were put on ice after reaction and 100 microL supernatant was transferred to a microtiter plate. OD595 was read as a measure of pullulanase. For each sample the reaction was performed triplicate.

Results for different ratio (the amount of amylopullulanase was kept at 10 micro g for each reaction). The activity of the 10 micro g amylopullulanase alone was set to 100%.

Ratio profile for 50° C.

Sample ratio X47 domain 1 1 1 1 2 5 10 Thermococcus 10 5 2 1 1 1 1 hydrothermalis amylopullulanase Relative activity (%) 95 102 152 105 120 134 100

Ratio profile for 75° C.

Sample ratio X47 domain 1 1 1 1 2 5 10 Thermococcus 10 5 2 1 1 1 1 hydrothermalis amylopullulanase Relative activity (%) 97 90 89 127 133 109 89

Temperature Profile

10 micro g pullulanase from Thermococcus hydrothermalis (P6VK), 20 micro g X47, 100 microL 0.4% AZCL-HE-pullulan and 150 microL buffer at pH 4.5 were mixed and incubated at corresponding temperature for 50 min with shaking at 900 rpm. The tubes were put on ice after reaction and 100 microL supernatant was transferred to a microtiter plate. OD595 was read as a measure of pullulanase. For each sample the reaction was performed triplicate.

Temperature 50° C. 60° C. 70° C. 80° C. Relative activity 16 20 33 100 (%)

pH Profile

pH buffers: 100 mM Succinic acid, HEPES, CHES, CAPSO, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100, pH adjusted to 3.5, 4.0, 4.5, 5.0, 6.0 7.0, and 8.0 with HCl and NaOH.

10 micro g pullulanase from Thermococcus hydrothermalis (P6VK), 20 micro g X47, 100 microL 0.4% AZCL-HE-pullulan (in MilliQ) and 150 microL 50 mM pH buffer (3.5-8) were mixed and incubated at 50° C. for 30 min with shaking at 900 rpm. 100 microL supernatant was transferred to a new microtiter plate. OD595 was read as a measure of pullulanase activity. For each enzyme the reaction was performed triplicate.

PH 3.5 4 4.5 5 6 7 8 Relative activity (%) 13 36 100 66 55 57 57 Boosting Test with Alpha-Amylase

1 micro g of Subulispora alpha-amylase disclosed as SEQ ID NO: 2 in WO 2009/140504 (Novozymes); different amounts of X47, 100 microL 0.4% AZCL-HE-amylose (Megazyme International Ireland Ltd.) at pH 4.5, and different amount of milliQ water (to give the same final volume for different ratio) were mixed and incubated at 37° C. for 30 min with shaking at 900 rpm. The tubes were put on ice after reaction and 100 microL supernatant was transferred to a microtiter plate. OD595 was read as a measure of pullulanase. For each sample the reaction was performed triplicate.

Results for different ratio (the amount of alpha-amylase was kept at 1 micro g for each reaction).

The activity of the 1 micro g alpha-amylase alone was set to 100%.

Sample ratio X47 Domain 1 1 1 1 2 5 10 Amylase 10 5 2 1 1 1 1 Relative activity (%) 99 98 95 125 89 83 127

Example 13

Ethanol Production During Pullulanase in Combination with Protease

The full length pullulanase from Thermococcus hydrothermalis (Pullulanase H expressed in Pichia pastoris) was added into the liquefaction of corn in combination with Protease A and the Alpha-Amylase SC. The liquefactions were done at either pH 5.4, 30-32% dry solids, 85° C. in a water bath for two hours. The enzyme doses were 1, 5 or 50 micro g enzyme protein/gram dry solids for the pullulanase, 50 micro g enzyme protein/gram dry solids for the protease and 0.02% w/w corn for the Alpha-Amylase SC. The liquefied mashes were fermented with Glucoamyalase SF at a dose of 0.5 AGU/gram dry solids for 54 hours at 32° C. Table 1 present the average ethanol yields (in grams per liter) and standard deviations as quantified by HPLC.

The dose of Protease A in all cases was 50 micro g EP/g DS. The Pullulanase H dose was 1, 5 and 50 micro g EP/g DS. The control is the Alpha-Amylase SC alone without further enzyme additions. The pH of this liquefaction was 5.4 for all samples.

TABLE 1 Average ethanol yields (grams/liter) with standard deviations. Ethanol (Average ± standard deviation) Treatment Grams/Liter pH 5.4 Alpha-Amylase SC (control) 121.30 ± 0.22 Pullulanase H (1 micro g EP/g DS) + 121.52 ± 0.02 Protease A (50 micro g EP/g DS) Pullulanase H (5 micro g EP/g DS) + 122.30 ± 0.25 Metalloprotease A (50 micro g EP/g DS) Pullulanase H (50 micro g EP/g DS) + 122.65 ± 0.60 Protease A (50 micro g EP/g DS)

The present invention is further described in the following numbered paragraphs:

-   [1] A pullulanase variant of a parent pullulanase belonging to     family GH57 and comprising an X47 domain, wherein the pullulanase     variant is truncated after the X47 domain. -   [2] The pullulanase variant of paragraph 2, wherein the parent     pullulanase is obtained from a strain from the genus Thermococcus,     including Thermococcus sp. AM4, Thermococcus sp. HJ21, Thermococcus     barophilus; Thermococcus gammatolerans; Thermococcus kodakarensis;     Thermococcus litoralis, preferably the one shown in SEQ ID NO: 4;     Thermococcus hydrothermalis, preferably the one shown in SEQ ID NO:     2; Thermococcus onnurineus; or is obtained from a strain of the     genus Pyrococcus, such as Pyrococcus abyssi and Pyrococcus furiosus. -   [3] The pullulanase variant of paragraph 1 or 2, wherein the parent     pullulanase is one of the pullulanases shown in SEQ ID NO: 2 (T.     hydrothermalis), SEQ ID NO: 4 (T. litoralis), or the hybrid shown in     SEQ ID NO: 34, or a sequence having pullulanase activity being at     least 60% identical to SEQ ID NOs: 2, 4, or 34. -   [4] The pullulanase variant of any of paragraphs 1-3, wherein the     parent pullulanase has one or more (several) amino acids     substituted, deleted, and/or inserted compared to SEQ ID NOS: 2, 4     or 34, or in another parent pullulanase. -   [5] The pullulanase variant of any of paragraphs 1-4, wherein the     parent pullulanase has at least 70%, preferably at least 80%     preferably at least 85%, preferably at least 90%, at least 95%, at     least 96%, at least 97%, at least 98%, at least 99% identity to the     mature parent pullulanase of SEQ ID NOS: 2, 4, or 34. -   [6] The pullulanase variant of any of paragraphs 1-5, wherein the     pullulanase variant is at least 70%, preferably at least 80%,     preferably at least 85%, more preferably 90%, more preferably 95%,     more preferably 97%, at least 98%, at least 99% identical to the     mature parent pullulanase of SEQ ID NOS 2, 4, or 34. -   [7] The pullulanase variant of any of paragraphs 1-6, wherein the     pullulanase variant has pullulanase activity. -   [8] The pullulanase variant of any of paragraphs 1-7, further     wherein the total number of different amino acids in the parent     pullulanase or the pullulanase variant is fifteen, more preferably     fourteen, even more preferably thirteen, even more preferably     twelve, even more preferably eleven, even more preferably ten, even     more preferably nine, even more preferably eight, even more     preferably seven, even more preferably six, even more preferably     five, even more preferably four, even more preferably three, even     more preferably two, and most preferably one. -   [9] The pullulanase variant of any of paragraphs 1-8, wherein the     truncation is in the DUF2223a domain or DUF2223b domain. -   [10] The pullulanase variant of any of paragraphs 1-8, wherein the     truncation is within 100 amino acids, preferably within 50 amino     acids, preferably within 20 amino acids after the end of the X47     domain. -   [11] The pullulanase variant of any of paragraphs 1-10, wherein the     variant is prepared from a parent pullulanase belonging to family     GH57 which comprises an X47 domain, wherein the parent pullulanase     is one shown in SEQ ID NO: 2, 4 or 34, or another parent pullulanase     having at least 60% identity to SEQ ID NO: 2, 4 or 34, wherein the     pullulanase variant comprises or consists of:

a) an amino acid sequence having pullulanase activity;

-   -   i) having at least 60% identity to the sequence from amino acids         1-1009 of SEQ ID NO: 2, preferably to the sequence from amino         acids 1-782 of SEQ ID NO: 2; or     -   ii) having at least 60% identity to the sequence from amino         acids 1-988 of SEQ ID NO: 4, preferably to the sequence from         amino acids 1-781 of SEQ ID NO: 4;     -   iii) having at least 60% identity to the sequence from amino         acids 1-782 of SEQ ID NO: 34;

b) the parent pullulanase of SEQ ID NOs: 2, 4 or 34 truncated at a position after the X47 domain;

c) another parent pullulanase having at least 60% identity to SEQ ID NOs: 2 4, or 34 truncated in a position corresponding to the ones defined in a) or b);

d) a pullulanase variant defined in a), b) or c) having one or more (several) amino acids substituted, deleted, and/or inserted.

-   [12] The pullulanase variant of paragraph 11, wherein the parent     pullulanase is obtained from a strain from the genus Thermococcus,     including Thermococcus sp. AM4, Thermococcus sp. HJ21, Thermococcus     barophilus, Thermococcus gammatolerans, Thermococcus kodakarensis,     Thermococcus litoralis, preferably the one shown in SEQ ID NO: 4;     Thermococcus hydrothermalis; Thermococcus onnurineus; or obtained     from a strain of the genus Pyrococcus, such as Pyrococcus abyssi and     Pyrococcus furiosus. -   [13] The pullulanase variant of paragraph 11 or 12, wherein the     parent pullulanase has at least 70%, preferably at least 80%,     preferably at least 85%, preferably at least 90%, at least 95%, at     least 96%, at least 97%, at least 98%, at least 99% identity to the     mature pullulanase of SEQ ID NOs: 2 4, or 34. -   [14] The pullulanase variant of any of paragraphs 11-13, wherein the     pullulanase variant has at least 70%, preferably at least 80%,     preferably at least 85%, preferably at least 90%, at least 95%, at     least 96%, at least 97%, at least 98%, at least 99% identity to the     mature part of any of the pullulanases in SEQ ID NOs: 2, 4, or 34,     preferably the sequence from amino acids 1-1009 of SEQ ID NO: 2 or     from amino acids 1-988 in SEQ ID NO: 4 or the sequence from amino     acids 1-782 of SEQ ID NO: 34, preferably to the sequence from amino     acids 1-782 of SEQ ID NOS: 2 or from the sequence from amino acids     1-781 of SEQ ID NO: 4. -   [15] The pullulanase variant of any of paragraphs 1-14, wherein the     parent pullulanase is a wild-type pullulanase. -   [16] The pullulanase variant of any of paragraphs 11-15, wherein the     truncation is in the DUF2223a domain, such as the DUF2223a domain     located from positions 769-1009 in SEQ ID NO: 2, or positions     768-988 in SEQ ID NO: 4, or 769-782 in SEQ ID NO: 34, or in a     corresponding positions in another parent pullulanase. -   [17] The pullulanase variant of any of paragraphs 11-16, wherein the     truncation is between amino acids in positions 782-783 in SEQ ID NO:     2, which corresponds to positions between positions 781-782 in SEQ     ID NO: 4, or in corresponding positions in another parent     pullulanase. -   [18] The pullulanase variant of any of paragraphs 11-17, wherein the     truncation is within 100 amino acids, preferably 50 amino acids,     preferably 20 amino acids of the end of the X47 domain which ends at     position 768 in SEQ ID NO: 2, position 767 in SEQ ID NO: 4, and     position 768 in SEQ ID NO: 34, or a corresponding position in     another parent pullulanase. -   [19] The pullulanase variant of any of paragraphs 11-18, further     wherein the total number of different amino acids in the parent     pullulanase or the pullulanase variant is fifteen, more preferably     fourteen, even more preferably thirteen, even more preferably     twelve, even more preferably eleven, even more preferably ten, even     more preferably nine, even more preferably eight, even more     preferably seven, even more preferably six, even more preferably     five, even more preferably four, even more preferably three, even     more preferably two, and most preferably one. -   [20] The pullulanse variant of any of paragraphs 1-19, which     pullulanase variant has higher activity compared to the parent     pullulanase. -   [21] The pullulanase variant of any of paragraphs 15-20, wherein the     parent pullulanase is encoded by a nucleic acid sequence which     hybridizes under medium, more preferably high stringency conditions,     with the nucleic acid sequence of SEQ ID NOs: 1 or 3, or its     complementary strand. -   [22] An X47 domain. -   [23] The X47 domain of paragraph 22, wherein the domain is     obtainable from a strain of the genus Thermococcus, including     Thermococcus sp. AM4, Thermococcus sp. HJ21, Thermococcus     barophilus, Thermococcus gammatolerans, Thermococcus kodakarensis,     Thermococcus litoralis; Thermococcus hydrothermalis; Thermococcus     onnurineus; or obtained from a strain of the genus Pyrococcus, such     as Pyrococcus abyssi and Pyrococcus furiosus. -   [24] The X47 domain of paragraph 22 or 23 comprising or consisting     of the amino acid sequence 580-768 in SEQ ID NO: 2, amino acid     sequence 579-767 in SEQ ID NO: 4 or the amino acid sequence 580-768     in SEQ ID NO: 34, or the amino acid sequence 86-274 shown in SEQ ID     NO: 38, or corresponding position in another parent pullulanases. -   [25] The X47 domain of any of paragraphs 22-24 comprising or     consisting of the amino acid sequence 580-768 in SEQ ID NO: 2 or     amino acid sequence 579-767 in SEQ ID NO: 4 or the amino acid     sequence 580-768 in SEQ ID NO: 34, or the amino acid sequence 86-274     shown in SEQ ID NO: 38, or a corresponding positions in another     parent pullulanases determined by Hidden Markov Model (HMM) having a     score of at least 300, preferably a score of at least 350,     preferably a score of at least 400, preferably between 300-500, such     as between 380-450. -   [26] The X47 domain of any of paragraphs 22-25, having at least 60%     identity to amino acid sequence 580-768 in SEQ ID NO: 2 or amino     acid sequence 579-767 in SEQ ID NO: 4 or amino acid sequence 580-768     in SEQ ID NO: 34 or amino acid sequence 86-274 in SEQ ID NO: 38. -   [27] The X47 domain of any of paragraphs 22-26, wherein the X47 has     at least 70%, preferably at least 80%, preferably at least 85%,     preferably at least 90%, at least 95%, at least 96%, at least 97%,     at least 98%, at least 99% identity to amino acid sequence 580-768     in SEQ ID NO: 2 or to amino acid sequence 579-767 in SEQ ID NO: 4 or     amino acid sequence 580-768 in SEQ ID NO: 34. -   [28] An isolated polynucleotide comprising a nucleotide sequence     which encodes the pullulanase variant of any of paragraphs 1-21 or     X47 domain of paragraphs 22-27. -   [29] An isolated polynucleotide encoding a pullulanase variant of     any of paragraphs 1-21, or an X47 domain of paragraphs 22-27,     selected from the group consisting of:     -   i) a polynucleotide having at least 60%, preferably at least         70%, more preferably at least 80%, more preferably at least 90%,         more preferably at least 95%, at least 96%, preferably at least         97%, preferably at least 98%, preferably at least 99% identity         with the pullulanase variant coding part of SEQ ID NOS: 1 or 3,         or a complementary strand thereof;     -   ii) a polynucleotide having at least 60%, preferably at least         70%, more preferably at least 80%, more preferably at least 90%,         more preferably at least 95%, at least 96%, preferably at least         97%, preferably at least 98%, preferably at least 99% identity         with the X47 domain coding part of sequence SEQ ID NO: 1, 3, or         37, or a complementary strand thereof; and     -   iii) a polynucleotide which hybridizes under medium stringency,         preferably high stringency conditions with the pullulanase         variant or X47 domain coding part of SEQ ID NO: 1, 3, or 37, or         a complementary strand thereof. -   [30] A nucleic acid construct comprising the polynucleotide of     paragraphs 28 or 29 operably linked to one or more (several) control     sequences which direct the production of the polypeptide in an     expression host. -   [31] A recombinant expression vector comprising the nucleic acid     construct of paragraph 30. -   [32] A recombinant host cell comprising the nucleic acid construct     of paragraph 30 or the vector of paragraph 31. -   [33] A method for producing the pullulanase variant of any of     paragraphs 1-25 or an X47 domain of any of claims 7-10     comprising (a) cultivating the recombinant host cell of paragraph 32     under conditions conducive for production of the pullulanase variant     or X47 domain; and (b) recovering the pullulanase variantand/or X47     domain. -   [34] The method of paragraph 33, wherein the expression level of the     variant is the same or higher compared to the corresponding parent     pullulanase under the same conditions. -   [35] A preparation comprising a pullulanase variant having:

(a) a temperature optimum in the range between 65-100° C., preferably 70-90° C., especially 75-85° C.;

(b) a pH optimum in the range between pH 4-6, preferably 4.5-5.5 with AZCL-pullulan as substrate.

-   [36] A process for producing a fermentation product from     starch-containing material comprising the steps of:

(a) liquefying starch-containing material in the presence of an alpha-amylase and a family GH57 pullulanase;

(b) saccharifying the liquefied material obtained in step (a) using a carbohydrate-source generating enzyme;

(c) fermenting using a fermenting organism.

-   [37] The process of paragraph 36, wherein the GH57 pullulanase is     obtained from a strain from the genus Thermococcus, including     Thermococcus sp. AM4, Thermococcus sp. HJ21, Thermococcus     barophilus, Thermococcus gammatolerans, Thermococcus kodakarensis,     Thermococcus litoralis, preferably the one shown in SEQ ID NO: 4;     Thermococcus hydrothermalis; Thermococcus onnurineus; or obtained     from a strain of the genus Pyrococcus, such as Pyrococcus abyssi and     Pyrococcus furiosus or the pullulanase in SEQ ID NO: 34. -   [38] The process of paragraph 36 or 37, wherein the pullulanase has     at least 70%, preferably at least 80%, preferably at least 85%,     preferably at least 90%, at least 95%, at least 96%, at least 97%,     at least 98%, at least 99% identity to the mature parent     pullulanase, preferably the parent pullulanase shown in SEQ ID NOs:     2, 4 or 34. -   [39] The process of paragraph 37, wherein the family GH57     pullulanase is a pullulanase variant of any of paragraphs 1-21 -   [40] The process of any of paragraphs 36-39, further wherein a     protease, such as an acid fungal protease or a metalloprotease is     added before, during and/or after liquefaction. -   [41] The process of paragraph 40, wherein themetallo protease is     derived from a strain of the genus Thermoascus, preferably a strain     of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC     No. 0670. -   [42] The process of any of paragraphs 36-41, wherein the     alpha-amylase is a fungal alpha-amylase, preferably an acid fungal     alpha-amylase or a bacterial alpha-amylase. -   [43] The process of any of paragraphs 36-42, wherein the     alpha-amylase is a fungal alpha-amylase, preferably derived from the     genus Aspergillus, especially a strain of A. niger, A. oryzae, A.     awamori, or Aspergillus kawachii, or of the genus Rhizomucor,     preferably a strain the Rhizomucor pusillus, or the genus Meripilus,     preferably a strain of Meripilus giganteus. -   [44] The process of any of paragraphs 36-43, wherein the     alpha-amylase is one disclosed in Richardson et al., 2002, The     Journal of Biological Chemistry 277(29): 267501-26507 (Issue 19     July), preferably the one referred to as “BD5088”. -   [45] The process of any of paragraphs 36-44, wherein the     alpha-amylase is present in an amount of 0.001 to 10 AFAU/g DS,     preferably 0.01 to 5 AFAU/g DS, especially 0.3 to 2 AFAU/g DS or     0.001 to 1 FAU-F/g DS, preferably 0.01 to 1 FAU-F/g DS. -   [46] The process of any of paragraphs 36-45, wherein the     alpha-amylase is a bacterial alpha-amylase derived from a strain of     Bacillus, such as Bacillus stearothermophilus, in particular the     Bacillus stearothermophilus as disclosed in WO99/019467 as SEQ ID     No: 3 with the double deletion 1181+G182 and substitution N193F     and/or a hybrid alpha-amylase comprises 445 C-terminal amino acid     residues of the Bacillus licheniformis alpha-amylase (shown in SEQ     ID NO: 4 of WO 99/19467) and the 37 N-terminal amino acid residues     of the alpha-amylase derived from Bacillus amyloliguefaciens (shown     in SEQ ID NO: 5 of WO 99/19467), with the following substitution:     G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S (using the     Bacillus licheniformis numbering in SEQ ID NO: 4 of WO 99/19467). -   [47] The process of any of paragraphs 36-46, wherein the     alpha-amylase is dosed in an amount of 0.0005-5 KNU per g DS,     preferably 0.001-1 KNU per g DS, such as around 0.050 KNU per g DS. -   [48] The process of any of paragraphs 36-47, wherein the     carbohydrate-source generating enzyme is selected from the group     consisting of glucoamylase, alpha-glucosidase, maltogenic amylase,     and beta-amylase. -   [49] The process of any of paragraphs 36-48, wherein the     carbohydrase-source generating enzyme is glucoamylase and is present     in an amount of 0.001 to 10 AGU/g DS, preferably from 0.01 to 5     AGU/g DS, especially 0.1 to 0.5 AGU/g DS. -   [50] The process of any of paragraphs 36-49, wherein the     glucoamylase is derived from a strain of Aspergillus, preferably     Aspergillus niger or Aspergillus awamori, a strain of Talaromyces,     especially Talaromyces emersonii; or a strain of Athelia, especially     Athelia rolfsii; a strain of Trametes, preferably Trametes     cingulata; a strain of the genus Pachykytospora, preferably a strain     of Pachykytospora papyracea; or a strain of the genus Leucopaxillus,     preferably Leucopaxillus giganteus; or a strain of the genus     Peniophora, preferably a strain of the species Peniophora     rufomarginata; or a mixture thereof. -   [51] The process of any of paragraphs 36-50, wherein step (a) is     carried out at pH 4.-6, preferably at a pH from 4.5 to 5.5. -   [52] The process of any of paragraphs 36-51, wherein the     fermentation product is recovered after fermentation, preferably by     distillation. -   [53] The process of any of paragraphs 36-52, wherein the step (b)     and (c) are carried out sequentially or simultaneously (i.e., SSF     process). -   [54] The process of any of paragraphs 36-53, wherein the     fermentation product is an alcohol, preferably ethanol, especially     fuel ethanol, potable ethanol and/or industrial ethanol. -   [55] The process of any of paragraphs 36-54, wherein the     starch-containing starting material is whole grains. -   [56] The process of any of paragraphs 36-55, wherein the     starch-containing material is derived from corn, wheat, barley, rye,     milo, sago, cassava, manioc, tapioca, sorghum, rice or potatoes. -   [57] The process of any of paragraphs 36-56, wherein the fermenting     organism is a strain of Saccharomyces, preferably a strain of     Saccharomyces cerevisae. -   [58] The process of any of paragraphs 36-57, further comprising,     prior to the step (a), the steps of:

x) reducing the particle size of starch-containing material, preferably to a particle size to between 0.05 to 3.0 mm, preferably 0.1-0.5 mm, so at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90% of the starch-containing material fit through a sieve with a 0.05 to 3.0 mm screen, preferably 0.1-0.5 mm screen;

y) forming a slurry comprising the starch-containing material and water.

-   [59] The process of paragraph 58, wherein the slurry is heated to     above the gelatinization temperature. -   [60] The process of paragraph 55 or 56, wherein the slurry is     jet-cooked at a temperature between 95-140° C., preferably 105-125°     C., for 1-15 minutes, preferably for 3-10 minutes, especially around     5 minutes. -   [61] The use of a pullulanase variant or X47 domain of any one of     paragraphs 1-27 or preparation of paragraph 35, in a process of     producing sweeteners from starch. λ[62] The use of a pullulanase     variant or X47 domain of any one of paragraphs 1-27 or preparation     of paragraph 35, in a process of producing a fermentation product,     such as ethanol, from gelatinized and/or un-gelatinized starch. 

1-20. (canceled)
 21. A pullulanase variant of a parent pullulanase belonging to family GH57 and comprising an X47 domain, wherein the pullulanase variant is truncated after the X47 domain.
 22. The pullulanase variant of claim 21, wherein the parent pullulanase is a SEQ ID NO: 2 (T. hydrothermalis), SEQ ID NO: 4 (T. litoralis), SEQ ID NO: 34, or a sequence having pullulanase activity which is at least 60% identical to SEQ ID NO: 2, 4, or
 34. 23. The pullulanase variant of claim 21, wherein the parent pullulanase has at least 70%, preferably at least 80% preferably at least 85%, preferably at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the mature parent pullulanase of SEQ ID NOS: 2, 4, or
 34. 24. The pullulanase variant of claim 21, wherein the pullulanase variant is at least 70%, preferably at least 80%, preferably at least 85%, more preferably 90%, more preferably 95%, more preferably 97%, at least 98%, at least 99% identical to the mature parent pullulanase of SEQ ID NO: 2, 4, or
 34. 25. The pullulanase variant of claim 21, wherein the variant is prepared from a parent pullulanase belonging to family GH57 which comprises an X47 domain, wherein the parent pullulanase is one shown in SEQ ID NO: 2, 4 or 34, or another parent pullulanase having at least 60% identity to SEQ ID NO: 2, 4 or 34, wherein the pullulanase variant comprises or consists of: a) an amino acid sequence having pullulanase activity; i) having at least 60% identity to the sequence from amino acids 1-1009 of SEQ ID NO: 2, preferably to the sequence from amino acids 1-782 of SEQ ID NO: 2; or ii) having at least 60% identity to the sequence from amino acids 1-988 of SEQ ID NO: 4, preferably to the sequence from amino acids 1-781 of SEQ ID NO: 4; iii) having at least 60% identity to the sequence from amino acids 1-782 of SEQ ID NO: 34; b) the parent pullulanase of SEQ ID NOs: 2, 4 or 34 truncated at a position after the X47 domain; or c) another parent pullulanase having at least 60% identity to SEQ ID NO: 2 4, or 34 truncated in a position corresponding to the ones defined in a) or b).
 26. An X47 domain.
 27. The X47 domain of claim 26 comprising or consisting of the amino acid sequence 580-768 in SEQ ID NO: 2, amino acid sequence 579-767 in SEQ ID NO: 4 or the amino acid sequence 580-768 in SEQ ID NO: 34, or the amino acid sequence 86-274 shown in SEQ ID NO: 38, or corresponding position in another parent pullulanases.
 28. The X47 domain of claim 26, comprising or consisting of the amino acid sequence 580-768 in SEQ ID NO: 2 or amino acid sequence 579-767 in SEQ ID NO: 4 or the amino acid sequence 580-768 in SEQ ID NO: 34, or the amino acid sequence 86-274 shown in SEQ ID NO: 38, or a corresponding positions in another parent pullulanases determined by Hidden Markov Model (HMM) having a score of at least 300, preferably a score of at least 350, preferably a score of at least 400, preferably between 300-500, such as between 380-450.
 29. The X47 domain of claim 26, having at least 60% identity to amino acid sequence 580-768 in SEQ ID NO: 2 or amino acid sequence 579-767 in SEQ ID NO: 4 or amino acid sequence 580-768 in SEQ ID NO: 34 or amino acid sequence 86-274 in SEQ ID NO:
 38. 30. A process for producing a fermentation product from starch-containing material comprising the steps of: (a) liquefying starch-containing material in the presence of an alpha-amylase and a family GH57 pullulanase; (b) saccharifying the liquefied material obtained in step (a) using a carbohydrate-source generating enzyme; (c) fermenting using a fermenting organism.
 31. The process of claim 30, further comprising adding a protease, such as an acid fungal protease or a metalloprotease before, during and/or after liquefaction.
 32. An isolated polynucleotide comprising a nucleotide sequence which encodes the pullulanase variant of claim
 21. 33. An isolated polynucleotide encoding a pullulanase variant of claim 21: i) a polynucleotide having at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% identity with the pullulanase variant coding part of SEQ ID NOS: 1 or 3, or a complementary strand thereof; iii) a polynucleotide which hybridizes under medium stringency, preferably high stringency conditions with the pullulanase variant or X47 domain coding part of SEQ ID NO: 1 or 3 or a complementary strand thereof.
 34. A nucleic acid construct comprising the polynucleotide of claim 32 operably linked to one or more (several) control sequences which direct the production of the polypeptide in an expression host.
 35. A recombinant expression vector comprising the nucleic acid construct of claim
 34. 36. A recombinant host cell comprising the nucleic acid construct of claim
 34. 37. A method for producing a pullulanase variant, comprising (a) cultivating the recombinant host cell of claim 36 under conditions conducive for production of the pullulanase variant; and (b) recovering the pullulanase variant. 